The largemouth bronze gudgeon () is a dominant species and important economic fish in China, which is distributed in the upstream regions of the Yangtze River. However, it is currently classified as threatened due to a rapid decline in its population. Cryopreservation technology enables germplasm cells to be safely and effectively preserved for a long time, which will help with artificial reproduction (e.g., captive broodstock management and seedling cultivation). In addition, cryopreservation can assist in maintaining original genotypes for the conservation of threatened and endangered species. Many studies have suggested that due to the species specificity of fish sperm, the extender composition, cryoprotectant type, and cryoprotectant concentration are different during cryopreservation. More importantly, there is currently no research on sperm cryopreservation of the largemouth bronze gudgeon. In the present study, to establish a sperm cryopreservation method for the largemouth bronze gudgeon, a computer assisted sperm motion analysis system (CASA) was used to find a suitable sperm cryopreservation method, by comparing the effects of four extenders (D15, D20, L1, and D1) and three cryoprotectants, including dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and methanol (METH), at three concentrations (7.5%, 10%, and 12.5%; ) on sperm motility after thawing. Our procedures followed the two-step method of cooling the mixture and thawing the frozen semen in a 37℃ water bath for 12-15 s. The results showed that D1 and METH are suitable choices for the extender and cryoprotectant, respectively. The motility of the sperm reached (78.11±14.74)%, which showed no significant difference compared to fresh sperm (>0.05). In comparison with the other treatments, the 10% METH group performed better in terms of the average velocity of (50.28±12.46) μm/s, average linear velocity of (35.06±10.82) μm/s, and average path velocity of (39.44±12.46) μm/s, which are significantly different values compared to those of fresh sperm (<0.05). The fastest movement time and lifetime of the sperm were (8.67±1.15) s and (33.33±5.00) s, respectively. In summary, 10% METH as an antifreeze protective agent in D1 was the best cryoprotectant protocol for sperm and could be applied in practice. The results provide a feasible technique for sperm preservation of largemouth bronze gudgeon, which could assist in artificial breeding and species conservation of the species.