Sertoli cells play an important physiological role in the testis, as they support, nourish, and protect the germ cells. As protection of the developing spermatozoa is an emerging aspect of reproductive physiology, this study examined the expression pattern of innate immune related genes, including Avian β-Defensins (AvBDs), Toll-like receptors (TLRs) and cytokines and investigated the time course of an inflammatory response in rooster Sertoli cells triggered by exposure to the bacterial endotoxin lipopolysaccharide (LPS). Sertoli cells were isolated from 6-week old chicken, cultured in vitro and stimulated with 1μg/ml LPS at different time courses (0, 6, 12, 24 and 48 h). Expression analysis data revealed that all 10 members of the chicken TLR family, 9 members of the AvBD family, as well as 8 cytokine genes were expressed in Sertoli cells. Quantitative real-time PCR analysis revealed that LPS treatment resulted in a significant induction in the expression levels of 6 TLR, 6 AvBD and 4 cytokine genes, while 2 cytokine genes were down-regulated and 2 others genes were unchanged. The increasing IL-1beta production was confirmed in the conditioned medium. Furthermore, the phagocytosis of Sertoli cells was increased after LPS treatment. In conclusion, these findings provide evidence that Sertoli cells express innate immune related genes and respond directly to bacterial ligands. These genes represent an important component of the immune system which could be integrated to semen, and present a distinctive constituent of the protective repertoire of the testis to ascending infections.