TRPV1 (transient receptor potential vanilloid 1) is a key molecule involved in the development of peripheral sensitization, which is believed to be the peripheral mechanism of pathological pain. Phosphorylation/dephosphorylation regulation plays a critical role in TRPV1 sensitization/desensitization. Multiple protein kinases, such as protein kinase A, protein kinase Cε, CaMKII (calmodulin-dependent kinase II) and Cdk5 (cyclin-dependent kinase 5), contribute to this process. Here, we reported an alternative way of functional regulation of TRPV1 by Cdk5, independent of the direct phosphorylation of TRPV1. The main findings are as follows: (1) TRPV1 can associate with KIF13B (kinesin-3 family member 13B) FHA domain. Knock down of endogenous KIF13B expression resulted in a significant decrease of the surface expression of TRPV1 in primary cultured DRG (dorsla root ganglion) neurons. (2) In vitro kinase assays showed that Cdk5 phosphorylated the KIF13B-FHA domain at Threonine-506. (3) Inhibition of Cdk5 activity decreased threonine phosphorylation of the KIF13B-motor FHA domain and the number of TRPV1-KIF13B-motor FHA complexes in CHO cells stably expressing TRPV1 (TRPV1-CHO cells) transfected with GFP-KIF13B motor FHA domain plasmids. (4) Transmembrane TAT-T506 peptide inhibited TRPV1 surface expression in DRG neurons. (5) Intrathecal delivery of the TAT-T506 peptide inhibited TRPV1 sensitization after inflammation and alleviated heat hyperalgesia behaviors in CFA-induced inflammatory pain models in rats. Conclusions: Regulation of the membrane trafficking of TRPV1 by Cdk5 through phosphorylating KIF13B contributes to inflammatory heat hyperalgesia. TAT-T506 might be a potential candidate for analgesic drug development.