Nanosized titanium dioxide (Nano TiO₂) has been widely used in daily lives, medicine, industry, and caused the potential reproduction toxicity for animals and human, however, the underlying molecular mechanisms for the reproductive toxicity of nano TiO₂ are still largely unclear. In the present study, when primary cultured rat Sertoli cells (SCs) were exposed to nano TiO₂, cell injury and alterations in wingless related MMTV integration site (Wnt) pathway-related factors including Wnt1, Wnt3a, Wnt5a, Wnt11, β-catenin, and p-GSK-3β expression were investigated. The results suggested that nano TiO₂ could be translocated to cytoplasm or nucleus, and decreased cell viability, and impaired morphological structures of SCs, induced apoptosis and dead of primary cultured rat SCs. Furthermore, nano TiO₂-induced the toxicity of primary cultured rat SCs was associated with increased expression of Wnt1, Wnt3a, Wnt5a, Wnt11, and β-catenin and involved with reduced p-GSK-3β expression. Therefore, this implies that nano TiO₂-induced toxic effects on SCs may be associated with Wnt signaling pathways.