Post-translational modifications in TRPV1 (transient receptor potential vanilloid 1) play a critical role in channel activity. Phosphorylation of serine/threonine residues within the N- and C-termini of TRPV1 are implicated in receptor sensitization and activation. Conversely, TRPV1 desensitization occurs via a calcium-dependent mechanism and leads to receptor de-phosphorylation. Importantly, we recently demonstrated that TRPV1 association with β-arrestin-2 is critical to receptor desensitization via its ability to scaffold the phosphodiesterase PDE4D5 to the receptor, regulating TRPV1 phosphorylation. In the present study, we demonstrate that phosphorylation of TRPV1 and β-arrestin-2 regulates this association at the membrane. Under serum-free media conditions, we observed a significant decrease in TRPV1 and β-arrestin-2 association in transfected CHO (Chinese-hamster ovary) cells. Pharmacological activation of the kinases PKA (protein kinase A) and PKC (protein kinase C) led to a robust increase in TRPV1 and β-arrestin-2 association, whereas inhibition of PKA and PKC decreased association. Previously, we identified potential PKA residues (Ser(116), Thr(370)) in the N-terminus of TRPV1 modulated by β-arrestin-2. In the present study we reveal that the phosphorylation status of Thr(370) dictates the β-arrestin-2 and TRPV1 association. Furthermore, we demonstrate that CK2 (casein kinase 2)-mediated phosphorylation of β-arrestin-2 at Thr(382) is critical for its association with TRPV1. Taken together, the findings of the present study suggest that phosphorylation controls the association of TRPV1 with β-arrestin-2.