Toll-like receptors (TLRs) play crucial roles in mediating innate and adaptive immunity. Sertoli cells create a microenvironment that protects seminiferous tubules from autoantigens and invading pathogens. Here, we examined the expression and potential function of TLR family in mouse Sertoli cells. RT-PCR, Western bloting and flow cytometry were used to analyze gene expression. Immunofluorescence staining was used to determine activation of NF-B. ELISA was used to detect secreted cytokines in culture medium. The phagocytosis assay was performed by ORO staining for lipid droplets. We demonstrated that TLR2, TLR3, TLR4 and TLR5 are highly expressed, TLR6, TLR7 and TLR13 are expressed at relatively low level, and TLR1, TLR8, TLR9, TLR11, TLR12 are not detected in mouse Sertoli cells. We focused our study on the roles of TLR2-TLR5 in Sertoli cells. Our data indicated that the TLR2-TLR5 can be activated by their ligands in mouse Sertoli cells and subsequently increase expression of the inflammatory cytokines IL-1, IL-6, IFN and IFN. The augmented expression of the cytokines might be induced by activation of NF-B. Notably, activation of TLR3 by its ligand, poly (I:C), specifically promoted phagocytosis of apoptotic spermatogenic cells by Sertoli cells. The TLR-induced Sertoli cell phagocytosis was found to be associated with the up-regulation of scavenger receptors. The results suggest that TLRs expressed in mouse Sertoli cells may play roles in defense against invasion of allo- and auto-antigens in the seminiferous tubules.