Rooster infertility is a major concern in the poultry industry and protection of the male reproductive organs from pathogens is an essential aspect of reproductive physiology. During the last years, research on antimicrobial protection has elucidated the critical role of the antimicrobial peptides avian β-defensins (AvBDs) in the innate immunity in chickens. AvBDs have been reported to be expressed in the hen reproductive organs, providing protection against microbial pathogens including Salmonella Enteritidis (SE). However, mechanisms of antimicrobial protection of rooster reproductive organs and especially the testis, mediated by AvBDs are poorly understood. The aim of this study was to investigate the expression of the complete family of the 14 AvBD genes, in the rooster testis in vivo, to determine whether sexual maturation affects their testicular mRNA abundance and to investigate whether SE infection alters their expression. Expression analysis revealed that 9 members of the AvBD family, namely AvBD1, 2, 4, 5, 6, 9, 10, 12 and 14 were expressed in the testis. Quantitative real-time PCR analysis revealed that the mRNA abundance of three AvBDs was up regulated and of three AvBDs was down regulated with respect to sexual maturation. In addition, SE infection resulted in a significant induction of AvBD4, 10, 12 and 14 in the testis of sexually mature roosters. These findings provide strong evidence to suggest that an AvBD-mediated immune response mechanism exists in the rooster testis providing protection against bacterial pathogens including Salmonella species.