ABSTRACT: Background: In the life history of Daphnia, the reproductive mode of parthenogenesis and sexual reproduction alternate in aquatic ecosystem, which are often affected by environmental and genetic factor. Although several functional genes on reproductive transition of Daphnia had been determined, molecular mechanism on the reproductive mode of Daphnia are still not known well, including differentially expressed genes in different developmental stages. Results: In this study, four developmental stages, juvenile female (JF), parthenogenetic female (PF), sexual female (SF) and male (M), of D. similoides sinensis were performed for transcriptome sequence, and candidate genes related to male sex determination were screened. A total of 110437 transcripts were obtained and assembled into 22996 unigenes. In the four developmental stages (JF, PF, SF and M), the number of unique unigenes is respectively 2863, 445, 437 and 586, and the number of common unigenes is 9708. The differentially expressed genes (DEGs) between male and other three female stages (M vs JF, M vs PF and M vs SF) were obtained. The GO gene enrichment analysis showed that the up-regulated genes in male were mainly enriched in hydrolase activity and peptidase activity. Thirty-six candidate genes related to male sex determination in male were significantly higher expression than those in the other three stages, including one Doublesex (Dsx) gene, one laminin gene, five trypsin genes and one serine protease genes, and one chitin synthase gene and two chitinase genes. In addition, in D. similoides sinensis male, the relative expression levels of two genes (Dsx1, antp) related to male sex determination observed in other Daphnia species were also significantly higher than those in other developmental stages. Conclusions: Our results showed that thirty-six candidate genes may involve in sex differetiation of D. similoides sinensis male, and it will provide a reference for further exploring the functional genes related to sex determination mechanism in Daphnia species. Moreover, according to previous investigations, we thought that the expression level of functional genes may be related to the development stage of organisms, and may be also affected by different Daphnia species.