Prostate cancer (PCa) is a malignant tumor with an extremely high prevalence. Doxorubicin is the first-line clinical treatment for castration-resistant PCa. Clinically, relapse is almost inevitable due to the cancer cells' increasing resistance to doxorubicin. Our previous studies have revealed that retinoic acid-related orphan nuclear receptor γ (RORγ) is a key protein for cancer progression and a promising target for PCa therapy. Though, RORγ's role and mechanism in doxorubicin-resistant PCa remain unclear. To study the mechanism of doxorubicin resistance, we generated a doxorubicin-resistant PCa cell line C4-2B (C4-2B DoxR) in this study, by culturing cells in an increasing doxorubicin concentration. Here, we show that RORγ expression was upregulated in C4-2B DoxR cells compared with that in normal C4-2B cells. The RORγ-stably-overexpressing PCa cell line constructed by lentiviral transfection showed an obvious improvement in doxorubicin resistance and a trend toward castration resistance. Furthermore, RORγ-specific small molecule inhibitors XY018, GSK805, and SR2211 can significantly inhibit the proliferation of C4-2B DoxR cells and promote their apoptosis. Collectively, these results have demonstrated the correlation between the upregulation of RORγ and the development of PCa's doxorubicin resistance, thus providing new ideas for solving the problem of chemotherapy drug resistance in PCa. © 2020 International Federation for Cell Biology.