Carnitine/organic cation transporter 2 (OCTN2) is localized at the basolateral membrane of epididymal epithelial cells, and mainly serves to reabsorb carnitine as an essential factor for sperm maturation; however, its functional features in epididymal epithelial cells have remained unclear. We isolated primary epididymal epithelial cells from rat epididymides and verified their phenotype by detecting the presence of cytokeratin-19 (CK-19, an epithelial cell marker) and the absence vimentin (an interstitial cell marker). We found that cultured epididymal epithelial cells isolated from rat epididymides expressed high levels of CK-19 but barely expressed vimentin. Gain-of-function assays, which included the CCK-8 assay and EdU flow cytometry assay, indicated that overexpression of OCTN2 significantly promoted epididymal epithelial cell growth and proliferation. Moreover, forced expression of OCTN2 inhibited the cell apoptosis process, and at the same time increased expression of the pro-apoptosis factor BAX, and decreased expression of the anti-apoptosis factors BCL-2 and Survivin. Furthermore, we also found that OCTN2 overexpression dramatically increased the levels of biomarkers associated with spermatogenesis, including azoospermia-like (DAZL), phosphoglycerate kinase 2 (PGK2), and protamine 2 (PRM2). These results demonstrate that OCTN2 plays a positive role in epididymal epithelial cells, and might be useful in the clinical treatment of male infertility by serving as a key regulatory factor.