Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.