Little is known about sex determination and differentiation in the Chinese soft-shelled turtle Pelodiscus sinensis. R-Spondin 1 (RSPO1), a candidate sex-determining gene, is an important regulator of ovarian differentiation in animals. Exogenous drugs can affect sex differentiation. In this study we cloned the RSPO1 gene from P. sinensis (psRSPO1) and analysed its expression profile. The psRSPO1 gene exhibited sequence identity with RSPO1 genes from other species. RSPO1 protein-based phylogenetic analysis showed that psRSPO1 in P. sinensis is closely related to RSPO1 proteins from other turtles. psRSPO1 showed abundant expression in adult brain and gonads, with higher levels in females than males. We also evaluated the effects of three finaconcentration of 2.5, 5.0 and 10mgmL-1 exogenous oestradiol (E2) and aromatase inhibitor (letrozole) on the expression of psRSPO1, external embryo morphology, growth status of embryos and the sex ratio when the drugs were injected to eggs during incubation. The expression of psRSPO1 was upregulated and downregulated by exogenous oestradiol and letrozole respectively, despite inconsistent expression trends at different embryo development times. External embryo morphology, growth status and sex ratio were affected by both exogenous oestradiol and the aromatase inhibitor. Feminisation was induced by oestradiol, but inhibited by letrozole. These results will contribute to studies of the potential molecular mechanisms underlying sex differentiation and sex control in the Chinese soft-shelled turtle.