Estrogens play a crucial role in the normal function of postnatal ovaries and testes in vertebrates. Estrogens have been reported to induce proliferation of spermatogonium stem cells and spermatogonia in both birds and mammals. Previous studies have showed that estrogens bind either to classical intracellular estrogen receptors (Esr1 and Esr2) or to a membrane estrogen receptor such as G protein-coupled estrogen receptor (Gper) and can therefore trigger both genomic or non-genomic signaling pathways. In comparison with the genomic pathway, non-genomic signaling occurs rapidly (within seconds to minutes) and involves the production of secondary messengers, the activation of protein kinases, and the modulation of ion-channels. Gper is a 7-transmembrane protein that belongs to the G protein-coupled receptor (Gpcr) superfamily, the members of which participate in various endocrine and metabolic processes. Gper is widely found in vertebrates such as mammals, birds, reptiles, and fish and is involved in many signaling pathways regulating cell and nervous system repair and other important physiological functions. Gper is very important in germ cell proliferation, in females in particular. is also expressed in germ cells in males, but little information on the role of in gonadal reproduction is available. The Chinese soft-shelled turtle () is a member of the Reptilia family of Chelonia Trionychidae and is an economically important aquaculture species in China. To improve the economic benefits of , it is important to study the characteristics and the mechanism of gonadal differentiation and reproduction of this species. However, limited studies on the reproduction of the turtle exist. The aims of this study were to identify the sequence information and function of Gper in the male turtle. The full-length cDNA sequence was cloned using rapid amplification of cDNA ends technology. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression level of mRNA in different tissues and different developmental stages of the gonad. Finally, the role of was analyzed by injecting letrozole and G-15 to inhibit aromatizing enzyme and Gper was 2023 bp long, including 705 bp at the 5'-UTR, 241 bp at the 3'-UTR, and a 1077 bp open reading frame (ORF) encoding a peptide of 358 amino acids. One exon of the ORF was the same as other reptiles. The putative peptide contained 7-transmembrane domains and an Asp-Arg-Tyr (DRY) structure, and Gper therefore belongs to the Rhodopsin subfamily. Multiple sequence comparisons of vertebrate Gper proteins indicated that Gper has the highest similarity with the corresponding protein in Chelonia while it has the lowest similarity with the corresponding protein in fish. Quantitative real-time PCR detected , and the maximum level was detected in the brain, followed by the ovary. Moreover, under different incubation temperatures, expression in the critical stage of gonadal differentiation showed the same trend:the highest expression level was found in stage 16 of embryonic development, and significantly decreased with gonadal differentiation. After injection of letrozole and G-15, the expression of was significantly decreased in the letrozole treatment group, but the expression of 2 were significantly increased. After inhibiting Gper protein activity, the expression of 2 obviously increased. More than that, the expression of genes related to spermatogenesis and apoptosis significantly increased after G-15 treatment. From the above, the role of Gper in the testes of is likely to be involved in early gonadal differentiation and regulates the proliferation of male germ cells. This study provides new insights into the role of Gper in the gonads of