In this study, a full-length 2558 bp Sp-Sox14 cDNA sequence was obtained from Scylla paramamosain gonadal transcriptome database. The encoded protein consists of 427 amino acids and includes an HMG box domain. Phylogenetic tree analysis showed that Sp-Sox14 was closely clustered with Sox14 of other arthropods. Quantitative real-time PCR displayed that Sp-Sox14 had different expression levels in different tissues from mature male and female crabs with the highest expression in ovary and the heart of male crabs. During embryogenesis, Sp-Sox14 expression was the highest in eye-pigment formation stage. During the different stages of ovarian development, Sp-Sox14 was expressed at the highest level in previtellogenesis stage (O2) and which was significantly higher than other stages. During the testis development, the expression level of Sp-Sox14 was higher in mature sperm stage (T3) than in spermatocyte stage (T1) and spermatid stage (T2). It was speculated that Sp-Sox14 was involved in the early development of ovary and the process of sperm maturation. The whole mount in situ hybridization (WISH) results indicated that the positive signals of Sp-Sox14 were mainly distributed in the head and near the maxillipede at eye-pigment formation stage, and near the compound eyes at prehatching stage. A small number of Sp-Sox14 positive signals were still present in the head and near the appendages at hatching stage. The results of WISH implied that Sp-Sox14 was involved in the formation of the nervous organ as well as related to the occurrence of somite appendages.