The role of WNT/β-catenin-signaling pathway is critical in mouse Sertoli cell maturation and tumorigenesis. This study aims to examine the effects of WNT/β-catenin signaling on the cultured adult human Sertoli cells and the underlying molecular mechanisms. Glycogen synthase kinase 3β (GSK-3β) inhibitors, SB216763 and lithium chloride (LiCl), were used to activate WNT/β-catenin-signaling pathway. 5-Bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry were used to analyze the proliferation and cell cycle of cultured human Sertoli cells, respectively. C-myc expression was accessed by immunofluorescence, real-time polymerase chain reaction and Western blot. The effects of c-myc on Sertoli cell proliferation were investigated by RNA interference technology and BrdU incorporation assay. The results showed activation of WNT/β-catenin signaling stimulated human Sertoli cell proliferation. Obvious increases in c-myc messenger RNA and protein expression were observed after SB216763 and LiCl treatments. Knockdown of c-myc expression attenuated the ability of WNT/β-catenin signaling to stimulate the proliferation of human Sertoli cells. WNT/β-catenin signaling enhances human Sertoli cell proliferation via upregulation of c-myc expression.