Transient receptor potential vanilloid 1 (TRPV1) is a molecular sensor of noxious heat and capsaicin. Its channel activity can be modulated by several mechanisms. Here we identify a membrane protein, Pirt, as a regulator of TRPV1. Pirt is expressed in most nociceptive neurons in the dorsal root ganglia (DRG) including TRPV1-positive cells. Pirt null mice show impaired responsiveness to noxious heat and capsaicin. Noxious heat- and capsaicin-sensitive currents in Pirt-deficient DRG neurons are significantly attenuated. Heterologous expression of Pirt strongly enhances TRPV1-mediated currents. Furthermore, the C terminus of Pirt binds to TRPV1 and several phosphoinositides, including phosphatidylinositol-4,5-bisphosphate (PIP2), and can potentiate TRPV1. The PIP2 binding is dependent on the cluster of basic residues in the Pirt C terminus and is crucial for Pirt regulation of TRPV1. Importantly, the enhancement of TRPV1 by PIP2 requires Pirt. Therefore, Pirt is a key component of the TRPV1 complex and positively regulates TRPV1 activity.