Prostate cancer (PC) is a major health problem in adult males. TRPM8, a cationic TRP channel activated by cooling and menthol is upregulated in PC. However, the precise role of TRPM8 in PC is still unclear. Some studies hypothesized that TRPM8-mediated transmembrane Ca(2+) fluxes play a key role in cellular proliferation of PC cells. In contrast, other findings suggest that high TRPM8 levels may reduce the metastatic potential of PC cells. A detailed understanding of the response of TRPM8 channels to pharmacological modulators of their activity is relevant when considering potential therapies, targeting this ion channel to treat PC. We characterized the pharmacological and functional properties of native TRPM8 channels in four human prostate cell lines, PNT1A, LNCaP, DU145, and PC3, commonly used as experimental models of PC. PNT1A is a non-tumoral prostate cell line while the other three correspond to different stages of PC. Here, we show that cold- and agonist-evoked [Ca(2+)](i) responses in PC cells are much less sensitive to well-characterized agonists (menthol and icilin) and antagonists (BCTC, clotrimazole, and DD01050) of TRPM8 channels, compared to TRPM8 channels in other tissues, suggesting a different molecular composition and/or spatial organization. In addition, the forced overexpression of human TRPM8 facilitated the trafficking of TRPM8 channels residing in the endoplasmic reticulum to the plasma membrane, leading to a marked potentiation in the efficacy of the different blockers. These results predict that blockers of canonical TRPM8 channels may be less effective in halting proliferation of PC cells than expected.