Spermatogenesis relies upon communication between the developing germ cells and their supporting Sertoli cells involving inflammatory signalling pathways mediated by Toll-like receptors (TLRs). We have discovered that spermatogenic cells can activate inflammatory signalling through TLR2 and TLR4 via the intracellular adaptor protein, MyD88. Examination of MyD88-deficient mutant mice revealed that, although testis weights were 43% larger prior to puberty (at 16 days of age), by 6 months of age, testis weights were 8% lower in MyD88-/- mice compared with their MyD88+/+ siblings. While most seminiferous tubules appeared normal in the MyD88-/- mice, some tubules contained degenerating germ cells. TUNEL assay indicated a 2-fold increase of apoptotic germ cells in MyD88-/- testes, and 1.7 times more seminiferous tubules containing apoptotic germ cells, compared to control testes, consistent with the progressive decline in testis weight. Paradoxically, qRT-PCR on total testis RNA from 6-month-old mice revealed a significant upregulation of TLR4, and the inflammatory cytokines, interleukin (IL)-1a, IL-1b, tumour necrosis factor (TNF) and activin B (Inhbb), but a significant downregulation in expression of the MyD88-independent adapter protein, TRIF. Significantly, a 13-fold increase in expression of the alternative activating receptor, TREM-1, a receptor that interacts with TLR2 and TLR4 during inflammation and amplifies downstream inflammatory pathways, was observed in the MyD88-/- mice. These data indicate that the absence of MyD88 causes progressive spermatogenic disruption, accompanied by increased inflammatory gene expression. These observations suggest that MyD88 plays a significant role in regulating spermatogenesis, but the absence of MyD88 in the Sertoli cell may enhance compensatory responses that limit the potential damage by activating an inflammation amplifying pathway utilising TREM-1. In conclusion, this study has uncovered novel communication networks in the seminiferous epithelium that involve inflammatory signalling pathways and may be linked to testicular dysfunction.