The half-smooth tongue sole, Cynoglossus semilaevis, is an increasingly important marine flatfish of potentially significant aquaculture value in China. It exhibits prominent sexually dimorphic growth in which females grow two to three times faster and larger than males. The growth arrest and DNA-damage-inducible protein 45 gamma (Gadd45g) plays a major role in embryonic development and sex determination. We evaluated the physiological function of a homologous Gadd45g gene on sex determination in C. semilaevis. Using RACE, we obtained a Gadd45g homologous gene from half-smooth tongue sole (named Gadd45g3). The full length Gadd45g3 was 1 147 bp, encoding 155 amino acid residues. A ribosomal_L7Ae domain was found in the sequence. Amino acid sequence analysis revealed that C. semilaevis Gadd45g3 shared 79%–88% identity with Gadd45g-like proteins in other fish and 58%–67% with Gadd45g in bony fish and higher vertebrates. The phylogenetic tree suggested that Gadd45g3 in C. semilaevis and Gadd45g-like in other fish were closely clustered in one branch because of their high similarity. Gadd45g-like genes were homologous with Gadd45g and typical for fish. They were produced from the additional fish-specific whole genome duplication. The ratio of Ka to Ks was used to diagnose the pattern of sequence evolution. The Ka/Ks for all comparisons of Gadd45g3 with homologous genes in other species was <0.3 suggesting that Gadd45g homologous genes are highly conserved. However, the Ka/Ks ratio for Gadd45g3 and Gadd45g homologous genes in mammals was higher than the former comparisons, suggesting that the function of Gadd45g3 in C. semilaevis varies from that of Gadd45g in mice. FISH results revealed that the Gadd45g3 gene is located on the autosome. Gadd45g3 expression was highest in the brain of both males and females, suggesting it is involved in the neurological development of C. semilaevis. Gadd45g3 expression was significantly higher in the testis of mature males and neo-males than in the ovary. In 20-day-old male larva, the expression of Gadd45g3 was markedly higher in the gonad than in same-aged females, whereas there was no difference in the level of gonadal expression between sexes in 95-day-old larva. Our results suggest that Gadd45g3 is a gender related gene that is necessary for testis maturation. Additionally, Gadd45g3 is involved in sex determination prior to gonadal differentiation. Moreover, Gadd45g3 also plays an important role in the early development of the ovary and testis.