This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01–5 μg/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.