1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the most active form of vitamin D₃, and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1α,25(OH)₂D₃ for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1α,25(OH)₂D₃ in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1α,25(OH)₂D₃-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1α,25(OH)₂D₃ does not induce acute Ca²⁺ response, whereas menthol evokes an increase in [Ca²⁺]_i, which suggests that cross-talks of menthol-induced Ca²⁺ signaling with 1α,25(OH)₂D₃-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1α,25(OH)₂D₃ and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1α,25(OH)₂D₃-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1α,25(OH)₂D₃.