Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase implicated in development and diseases of the mammalian nervous system. We have previously reported that Cdk5 activity participates in the regulation of nociceptive signaling. The expression of Cdk5 and its activator p35 is upregulated in nociceptive neurons during inflammation. In p35 knockout mice (p35-/-), thermal nociceptive responses are decreased, and in p35-overexpressing transgenic mice (Tgp35), these responses are increased, indicating the important role of Cdk5 in the nociceptive process. Moreover, Cdk5 regulates mitogen-activated/extracellular signal-related protein kinase kinase1/2 (MEKK1/2) activity through a negative feedback loop during the peripheral inflammatory response. Here we report that Cdk5-mediated phosphorylation of the transient receptor potential vanilloid receptor (TRPV1) at threonine 407 can modulate agonist-induced calcium influx. TRPV1, a ligand-gated cation channel that is highly expressed in small-diameter sensory neurons, is activated by heat, protons, and capsaicin. Inhibition of Cdk5 activity in cultured DRG neurons resulted in a significant reduction of TRPV1-mediated calcium influx, and this effect could be reversed by restoring Cdk5 activity. Primary nociceptor-specific Cdk5 conditional knockout mice showed reduced TRPV1 phosphorylation, resulting in significant hypoalgesia. In conjunction with our previous reports, the present findings indicate an important role for Cdk5 activity in pain signaling and provide insights into a new molecular mechanism for the functional regulation of TRPV1. We believe that these findings will provide impetus to develop a new class of analgesics targeted for Cdk5-mediated phosphorylation to treat pain.