Given the poor outcome of patients with hormone-resistant (HR) prostate cancer, new strategies are needed to improve the current therapeutic regimens and/or develop novel treatments. We therefore aimed to provide a better understanding of the molecular mechanisms involved in the aggressive tumor behavior of HR and develop more rational anti-tumor therapies. Three HR prostate cancer cell lines (androgen receptor (AR)-positive LNCaP-HR and 22RV1-HR and AR-negative PC-3) were used. Changes in tumor behavior, treatment response, and related signaling in HR were investigated in vitro and in vivo. The results revealed that constitutional activation of STAT3 and overexpressions of DNMT1 were important in the transition of HR prostate cancer. Furthermore, DNMT1 expression was required for the maintenance of STAT3 activation. When DNMT1 activity in HR was blocked, aggressive tumor behavior and treatment resistance could be overcome, which was seen in both in vitro and in vivo experiments. The underlying changes associated with inhibited DNMT1 included less epithelial-mesenchymal changes, less invasion ability, slower tumor growth, and impaired DNA repair ability, which are independent of AR and p53 status. In conclusion, altered DNMT1 expression associated with activated STAT3 may be crucial in the transition of HR. Targeting DNMT1 could be a promising strategy for the treatment of HR prostate, as evidenced by inhibited tumor growth and enhanced radiosensitivity. These findings provide evidence for therapeutically targeting DNMT1 in HR prostate cancer.