Schizothorax prenanti is a rare fish in the upper reaches of the Yangtze River and used to be an important economic fish in its production area. However, in recent years, due to diseases and environmental changes, the number of Schizothorax prenanti in the wild has decreased sharply. Thus, there is an urgent need to protect this fish species. The establishment of Schizothorax cell lines is a cost-effective method to protect its germplasm resources. Since the establishment of the fish cell line RTG-2 in 1962, research on the establishment of fish cell lines has developed rapidly. A total of 783 cell lines have been established from various fish tissues, including embryos. However, thus far, reports on the establishment of cell lines from Schizothorax prenanti are lacking. The establishment of the Schizothorax prenanti cell line not only enables the protection of its germplasm resources but also facilitates research on its living environment toxicology, nutrition, immunology, and disease control without harming the existing population of this rare fish. Schizothorax prenanti can be infected with Streptococcus agalactiae in the natural environment, resulting in exophthalmos, surface hemorrhage, and neurological symptoms, as well as marked degeneration, necrosis, and inflammatory cell infiltration in various internal organs. Mucosal immunity is an important part of the immune system of this fish, wherein the gills, skin, and intestinal mucosa constitute the first line of defense against pathogenic microorganisms. Thus, the establishment of a stable growth of the gill filament cell line of Schizothorax prenanti will enable the study of immune mechanism and disease control of Schizothorax prenanti. The most commonly used media for fish cell culture are MEM and L-15, where L-15 is an amino acid-rich medium that does not require CO₂ to maintain the pH of the medium. Among the 280 newly established fish cell lines in the world from 2010 to 2020, 180 use L-15 medium, accounting for 64% of the total. In this study, L-15 was used as the basis to prepare the Schizothorax prenanti culture medium. The primary cell medium was based on L-15 medium supplemented with 5 ng/mL bFGF, 5 ng/mL EGF, 27.5 μmol/L beta-mercaptoethanol, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin B, and 20% fetal bovine serum (FBS). The cell subculture medium was based on L-15 supplemented with 5 ng/mL bFGF, 5 ng/mL EGF, 27.5 μmol/L beta-mercaptoethanol, and 15% fetal bovine serum. The cell cryopreservation solution was made of 50% L-15 medium supplemented with 40% FBS and 10% DMSO. Schizothorax prenanti with a body length of about 11 cm was taken and placed in purified water containing 500 U/mL penicillin, 500 μg/mL streptomycin, and 1.25/mL amphotericin B overnight. On the second day, anesthesia was used, and the fish was wiped with 75% alcohol and moved to an ultra-clean bench. Ophthalmic scissors were used to cut the gill arches and gill filaments, which were transferred to a Petri dish containing PBS. The gill filaments were cut along the gill arch with a scalpel before rinsing five times in PBS containing 500 U/mL penicillin, 500 μg/mL streptomycin, and 1.25 μg/mL amphotericin B, changing the PBS wash solution each time. After washing, the gill filaments were placed in a petri dish containing PBS. Then, a scalpel was used to cut the gill filaments into 1-2 mm³ sections. A disposable Pasteur pipette was used to transfer the gill filament sections into a 15-mL centrifuge tube, to which PBS washing solution was added three times to remove surface debris and mucus. Then, the tissue sections were inoculated into T25 culture flasks, and as little medium as possible to cover the tissue blocks was added, without causing the tissue blocks to float or move. The cells were placed in a 24 ℃ incubator for static culture, to which 1 mL of fresh primary medium was replaced after 48 h. Cell migration was observed daily. When the cells migrated from the tissue section, 3 mL of medium was added to continue the culture, which was changed every three days to remove non-adherent cells and impurities. In this study, a cell line derived from Schizothorax prenanti (SPG strain) was established for the first time. The cells were isolated from the gill filament tissue of Schizothorax prenanti with a uniform epithelium. These were then cultured with L-15 containing 15% serum and successfully passed to 55 passages in 15 months. Mitochondrial COI gene identification proved that this cell was derived from Schizothorax prenanti, wherein the number of chromosomes in the karyotype was 2n = 96. Cells stored in liquid nitrogen for 12 months can maintain over 75% viability after resuscitation. EGFP-N3 plasmid was transfected into SPG cells, and marked green fluorescent protein expression was observed. The viral analog poly(I:C) and Escherichia coli lipopolysaccharide LPS increased the expression of immune-related genes, such as IL-1β, IL-8, TNFa, and TLR22, indicating that the SPG strain can be used for immunological and toxicological studies. In addition, this cell line also plays an important role in germplasm preservation, exogenous protein expression, and in vitro biological studies of Schizothorax prenanti. Although over 783 fish cell lines have been established in the world today, many of these have not been fully explored. Furthermore, a major problem in the field of fish cell line development is that many of the developed cell lines are not deposited in user-friendly cell banks. To facilitate the acquisition of this cell by researchers, the cell line established in this study has been stored in the National Marine Aquatic Germplasm Bank (YSFRI-C-2020-SPG).