Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the β-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of β-defensins in the epididymis.