Sperm maturation in the epididymis is a tightly regulated process which involves secretion and addition of a variety of proteins onto the sperm surface. The molecular mechanisms governing these processes has gained interest in the last decade. In vitro model systems to study the role of epididymal proteins in sperm maturation and other physiological process are very important. Isolation of epididymal cells, culture of epididymal explants and generation of immortalized cells were standardized to be used as in vitro models to study epididymal function. However, isolation and maintenance of primary cultures of epididymal epithelial cells seems to be the best option because of its closeness to the in vivo conditions. Though structural and morphological characterization of primary cultures of epididymal epithelial cells were carried out, the same were not conducted at the molecular level. In this study, we isolated adult rat epididymal primary epithelial cells (EPECs) and characterized them for their purity and cell specific expression of molecular markers. Isolated EPECs exhibited normal cell morphology and were sub cultured and maintained up to 3 weeks. EPECs expressed the epithelial marker, E-cadherin and their purity was estimated to be 73% using flow cytometry. EPECs abundantly expressed CRISP1, Urp1a, Pate-F, Crisp1, Ar and Spag11e, markers of epididymal cells and were negative for Urp1b and Pate, markers negative for epididymis. Results of our study provide a systematic characterization of EPECs at the molecular level and thus a refinement to the previously reported characterization methods.