Vitamin D3 (VD3) is an effective adjunctive agent, enhancing the destructive effects of freezing in prostate cancer cryoablation studies. We investigated whether dose escalation of VD3 over several weeks, to model the increase in physiological VD3 levels if an oral supplement were prescribed, would be as or more effective than a single treatment 1 to 2 days prior to freezing. PC-3 cells in log phase growth to model aggressive, highly metabolically active prostate cancer were exposed to a gradually increasing dose of VD3 to a final dose of 80 nM over a 4-week period, maintained for 2 weeks at 80 nM, and then exposed to mild sublethal freezing temperatures. Results demonstrate that both acute 24-hour exposure to 80 nM VD3 and dose escalation resulted in enhanced cell death following freezing at -15°C or colder, with no significant differences between the 2 exposure regimes. Apoptotic analysis within the initial 24-hour period postfreeze revealed that VD3 treatment induced both caspase 8- and 9-mediated cell death, most notably in caspase 8 at 8-hour postfreeze. These results indicate that both the intrinsic and extrinsic apoptotic pathways are involved in VD3 sensitization prior to freezing. Additionally, both acute and gradual dose escalation regimes of VD3 exposure increase prostate cancer cell sensitivity to mild freezing. Importantly, this study expands upon previous reports and suggests that the combination of VD3 and freezing may offer an effective treatment for both slow growth and highly aggressive prostate cancers.