Abstract: Chinese soft-shelled turtle Pelodiscus sinensis is an important aquaculture species in China, the males have higher economic value due to their higher growth rate, larger body size and wider calipash compared to females. Understanding the mechanism of sex differentiation is crucial for the production of mono-sex male offspring. However, the molecular mechanism of sex determination and gonadal differentiation in Chinese soft-shelled turtle is still unclear. Sox8 plays a pivotal role in male sex differentiation and testis maintenance in vertebrates. Knockdown of Sox8 led to the feminization of ZZ Chinese soft-shelled turtle (Pelodiscus sinensis). In this study, we employed a lentivirus-mediated overexpression vector to elevate Sox8 expression in ZW gonads of Chinese soft-shelled turtles prior to sex differentiation. At stage 27, histological analysis and immunofluorescence staining for VASA, a germ cell marker, revealed that after the overexpression of Sox8, 50% of ZW gonads possessed a medulla with testis-specific seminiferous cords containing germ cells. qRT-PCR and immunofluorescence results showed that the expression of female-specific genes Foxl2 and Cyp19a1 was significantly reduced, while the expression of Dmrt1, Sox9 and Amh, which regulate testicular development, was up-regulated. One month after hatching, the gonad of a Sox8-overexpressed ZW individual showed distinct male characteristics: the gonad contained a well-developed medulla and degenerated cortex, the germ cells were located in the sex cords, and SOX9 and DMRT1 proteins were abundantly expressed in the medulla whereas FOXL2 and AROM were not expressed at all. The results indicate that overexpression of Sox8 induces male differentiation in the ZW turtle embryos, suggesting the role of Sox8 in male differentiation in Chinese soft-shelled turtle.