The pharmacological desensitization of receptors is a fundamental mechanism for regulating the activity of neuronal systems. The TRPA1 channel plays a key role in the processing of noxious information and can undergo functional desensitization by unknown mechanisms. Here we show that TRPA1 is desensitized by homologous (mustard oil; a TRPA1 agonist) and heterologous (capsaicin; a TRPV1 agonist) agonists via Ca2+-independent and Ca2+-dependent pathways, respectively, in sensory neurons. The pharmacological desensitization of TRPA1 by capsaicin and mustard oil is not influenced by activation of protein phosphatase 2B. However, it is regulated by phosphatidylinositol-4,5-bisphosphate depletion after capsaicin, but not mustard oil, application. Using a biosensor, we establish that capsaicin, unlike mustard oil, consistently activates phospholipase C in sensory neurons. We next demonstrate that TRPA1 desensitization is regulated by TRPV1, and it appears that mustard oil-induced TRPA1 internalization is prevented by coexpression with TRPV1 in a heterologous expression system and in sensory neurons. In conclusion, we propose novel mechanisms whereby TRPA1 activity undergoes pharmacological desensitization through multiple cellular pathways that are agonist dependent and modulated by TRPV1.