Cryotherapy has emerged as a primary treatment option for prostate cancer (CaP); however, incomplete ablation in the periphery of the cryogenic lesion can lead to recurrence. Accordingly, we investigated the use of a non-toxic adjunctive agent, vitamin D3 (VD3), with cryotherapy to sensitize CaP to low temperature-induced, non-ice rupture-related cell death. VD3 (calcitriol) has been identified as a possible adjunct in the treatment of cancer because of its antiproliferative and antitumorigenic properties. This study aimed to identify the cellular responses and molecular pathways activated when VD3 (calcitriol) is combined with cryotherapy in a murine CaP model. Single freeze-thaw events above -15 °C had little effect on cancer cell viability; however, pretreatment with calcitriol in conjunction with cryo significantly increased cell death. The -15 °C calcitriol combination increased cell death to 55% following a single freeze compared with negligible cell loss by freezing or calcitriol alone. Repeated cryo combination yielded 90% cell death compared with 65% in dual freeze-only cycles. Western blot analysis following calcitriol cryosensitization regimes confirmed the activation of apoptosis. Specifically, proapoptotic Bid and procaspase-3 were found to decrease at 1 h following combination treatment, indicating cleavage to the active forms. A parallel in vivo study confirmed the increased cell death when combining cryotherapy with calcitriol pretreatment. The development of an adjunctive therapy combining calcitriol and cryotherapy represents a potentially highly effective, less toxic, minimally invasive treatment option. These results suggest a role for calcitriol and cryo as a combinatorial treatment for CaP, with the potential for clinical translation.