The number of functional transient receptor potential vanilloid 1 (TRPV1) channels at the surface, especially at the peripheral terminals of primary sensory neurons, regulates heat sensitivity, and increased surface localization of TRPV1s contributes to heat hyperalgesia. However, the mechanisms for regulating TRPV1 surface localization are essentially unknown. Here, we show that cyclin-dependent kinase 5 (Cdk5), a new player in thermal pain sensation, positively regulates TRPV1 surface localization. Active Cdk5 was found to promote TRPV1 anterograde transport in vivo, suggesting a regulatory role of Cdk5 in TRPV1 membrane trafficking. TRPV1-containing vesicles bind to the forkhead-associated (FHA) domain of the KIF13B (kinesin-3 family member 13B) and are thus delivered to the cell surface. Overexpression of Cdk5 or its activator p35 promoted and inhibition of Cdk5 activity prevented the KIF13B-TRPV1 association, indicating that Cdk5 promotes TRPV1 anterograde transport by mediating the motor-cargo association. Cdk5 phosphorylates KIF13B at Thr-506, a residue located in the FHA domain. T506A mutation reduced the motor-cargo interaction and the cell-permeable TAT-T506 peptide, targeting to the Thr-506, decreased TRPV1 surface localization, demonstrating the essential role of Thr-506 phosphorylation in TRPV1 transport. Moreover, complete Freund's adjuvant (CFA) injection-induced activation of Cdk5 increased the anterograde transport of TRPV1s, contributing to the development and possibly the maintenance of heat hyperalgesia, whereas intrathecal delivery of the TAT-T506 peptide alleviated CFA-induced heat hyperalgesia in rats. Thus, Cdk5 regulation of TRPV1 membrane trafficking is a fundamental mechanism controlling the heat sensitivity of nociceptors, and moderate inhibition of Thr-506 phosphorylation during inflammation might be helpful for the treatment of inflammatory thermal pain.