Beta-defensins are small cationic peptides exhibiting broad spectrum antimicrobial properties. In humans, many beta-defensin genes are located within a cluster on chromosome 8p23. The sperm associated antigen 11 (SPAG11) gene is contained in this cluster and is unusual among the human beta-defensins due to its complex genomic structure and mRNA splicing pattern. Here we report the genomic organization of the Bos taurus SPAG11 gene located on chromosome 27q1.2, within a cluster of beta-defensin genes. The exon structures of the fused bovine SPAG11 gene and of the mosaic transcripts initiated at both A and B promoters were established, including identification of novel exons and transcripts not previously found in primate or rodent. Evolutionary analysis against primate, rodent, canine, and porcine orthologs was performed. In adult bulls SPAG11C, SPAG11E, and SPAG11U mRNAs were detected predominantly in the male reproductive tract, while SPAG11D transcript was detected in reproductive and nonreproductive tissues and SPAG11V and SPAG11W mRNAs were confined to testis. Differential expression of all six transcripts was observed in tissues from fetal and adult bulls, suggesting that similar mRNA splicing mechanisms govern SPAG11 gene expression during pre- and postnatal development. Immunolocalization of SPAG11C and SPAG11D/E was demonstrated in the epithelium of the epididymis and testis, and SPAG11D in association with epididymal spermatozoa. Recombinant full-length SPAG11D protein was strongly antibacterial, while the SPAG11E C-terminal peptide that contains the beta-defensin motif in its structure was somewhat less potent. Taken together, the results suggest that SPAG11 isoforms perform both immune and reproductive functions in cattle.