Abstract: In order to provide biological information for research on sex-determination mechanism in Pelodiscus sinensis, we here first attempted to clone and analyze the partial-length of Foxl2 cDNA. In addition, to address the differential expression of Foxl2 at genetic and physiological levels, both male and female sexes of Pelodiscus sinensis were treated with 10 mg/kg E₂ and 17α-methyltestosterone (MT), respectively; Foxl2 mRNA expression was quantitatively examined in the gonads after injection treatment at 6, 12, 24, and 48h, as well as 7 and 14d, respectively. Foxl2 (GenBank Accession No. KP734210) was achieved, belonging to Forkhead family of transcription factors that is involved in ovarian development and functional maintenance, as well as a 903 bp of open reading frame (ORF) encoding 300 amino acids. Multiple sequence alignment showed that Foxl2 contained typical FH-domain, and the most similar ortholog was Trachemys scripta, reaching up to 99%. Stability analysis of phylogenetic trees showed that Pelodiscus sinensis Foxl2 was clustered with reptile Foxl2 as a sub-branch, and was closely associated with Foxl2 from Chrysemys picta bellii. Results of quantitative reverse transcription PCR (RT-qPCR) indicated that, compared to the control group, E₂ significantly up-regulated Foxl2 mRNA repression in ovary after 24h (P<0.001), which in testis was significantly increased after 7 and 14d (P<0.001). MT treatment strongly and equally up-regulated expression levels ofFoxl2 mRNA in ovary and testis at 24h (P<0.001). These results suggested that E₂ and MT could up-regulate Foxl2 expression. Moreover, the sex differences in E₂ promoting Foxl2 expression is more significant than MT. It can be concluded that the present research contributes to better understanding of the functional role of Foxl2 and provide basic data for further study on how exogenous hormone mediates Foxl2 in Pelodiscus sinensis.