Inactivated hemagglutinating virus of Japan envelope (HVJ-E) has an antitumor effect and tumor immunity. We undertook an open-label, phase I, dose-escalation study in patients with castration-resistant prostate cancer (CRPC) to determine the safety and efficacy of intratumoral and s.c. injection of HVJ-E (GEN0101). Patients with CRPC, who were resistant to or unable to receive standard of care, were included. GEN0101 was injected directly into the prostate and s.c. in two 28-day treatment cycles. The primary end-points were to evaluate the safety and tolerability of GEN0101 and determine its recommended dose. The secondary end-points were to analyze the antitumor effect and tumor immunity. Three patients received 30 000 mNAU GEN0101 and 6 received 60 000 mNAU. There was no dose-limiting toxicity, and the recommended dose of GEN0101 was defined as 60 000 mNAU. Radiographically, 1 patient had stable disease and 2 had progressive disease in the low-dose group, whereas 5 patients had stable disease and 1 had progressive disease in the high-dose group. Three patients in the high-dose group showed reduction in lymph node metastasis. Prostate-specific antigen increase rates in the high-dose group were suppressed more than those in the low-dose group. Natural killer cell activity was enhanced in 2 patients of the low-dose group and in 5 patients in the high-dose group. In conclusion, intratumoral and s.c. injections of GEN0101 were well-tolerated and feasible to use. The study is registered with the UMIN Clinical Trials Registry (no. UMIN000017092).