Retinoic acid (RA), the active metabolite of vitamin A, is essential for male fertility. While RA’s role in testis germ cell development has been extensively studied, very little is known about whether RA is also important for epididymal function. Our laboratory previously discovered that blocking RA activity in the epididymis resulted in abnormal epithelial pathology and male infertility. This current study investigated the localization patterns of the RA receptors (RARα, RARβ, RARγ) and binding proteins (CRABP1 and CRABP2) in response to RA in epididymal epithelial cells using immunohistochemistry and immunocytochemistry. Our aim was to identify whether there are specific epididymal cell types and/or segments that actively signal using RA. Consistent with previous studies in the rat, RARα was present in the nucleus of all epithelial cells throughout the epididymis, with the strongest signal evident in the corpus. RARγ was present in the cytoplasm of all epithelial cells except the clear cells of the cauda, and RARβ was not detectable. CRABP1 was also not detected in the epididymis although CRABP2 displayed a distinct segment specific expression pattern, with positive principal cells only detected in the corpus and cauda regions. Analysis of staining patterns in the epididymal epithelial cell line, mECap18, revealed cytoplasmic signal for all RARs and CRABPs, although RARβ and RARγ were also detected in the nucleus. In the presence of RA, RARα and both CRABPs displayed a shift into the nucleus, in some cells as quickly as 20 mins. RARβ and RARγ showed no response to RA. This study has revealed that the RA signaling and binding proteins display distinct cellular localization patterns in the epididymis and show varying degrees of responsiveness to RA. Future studies will focus on identifying the key downstream targets of RA signalling in the epididymal epithelium.