The aim of this study was to determine the expression profiles of Toll-like receptors (TLR) in the testis and epididymis of rooster and whether the expression of IL-1β, IL-6, CXCLi2, and TLR-4 was affected by lipopolysaccharide (LPS), a TLR-4 ligand. Roosters were intravenously injected with LPS or phosphate-buffered saline. Testes and epididymis were collected before and after 3 or 6 h postinjection. Total RNA was isolated from those tissues and expression of TLR and proinflammatory cytokines was analyzed by reverse-transcription PCR and quantitative real-time PCR. Reverse-transcription PCR analysis revealed that 7 of the known 10 chicken TLR in the testis and 9 of 10 in the epididymis were expressed. Expression of TLR-4 was found in both tissues. Expression of TLR-4 was significantly upregulated by LPS in the testis but not in the epididymis. Injection with LPS upregulated the expression of IL-1β, IL-6, and CXCLi2 in the testis and epididymis by 3 to 6 h postinjection. However, injection with phosphate-buffered saline (control) did not affect their expression. These results suggest that proinflammatory cytokines and chemokine expression was upregulated by LPS probably through TLR-4 activation, and thus the reproductive tissues are comprehensively equipped to deal with a pathogenic insult.