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 blotting, and flow cytometry were used to analyze gene expression. Immunofluorescence staining was used to determine activation of nuclear factor-kappaB. ELISA was used to detect secreted cytokines in culture medium. The phagocytosis assay was performed by Oil Red O 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, and 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 TLR2-TLR5 can be activated by their ligands in mouse Sertoli cells and subsequently increase expression of the inflammatory cytokines IL-1alpha, IL-6, and interferon-alpha, and -beta. The augmented expression of the cytokines might be induced by activation of nuclear factor-kappaB. 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 autoantigens in the seminiferous tubules.