The transient receptor potential channel subtypes V1 (TRPV1) and A1 (TRPA1) play a critical role in the development of hyperalgesia in inflammatory pain models. Although several studies in animals and humans have demonstrated that capsaicin (CAP), a TRPV1-specific agonist, and mustard oil (MO), a TRPA1 agonist, evoke responses that undergo functional cross-desensitization in various models, the mechanisms mediating this phenomenon are largely unknown. In the present study, we evaluated the mechanisms underlying homologous and heterologous desensitization between CAP and MO responses in peripheral nociceptors using an in vitro neuropeptide release assay from acutely isolated rat hindpaw skin preparation and in vivo behavioral assessments. The pretreatment with CAP or MO significantly inhibited (50–60%) both CAP-and MO-evoked CGRP release indicating homologous and heterologous desensitization using this assay. Further studies evaluating the requirement of calcium in these phenomena revealed that homologous desensitization of CAP responses was calcium-dependent while homologous desensitization of MO responses was calcium-independent. Moreover, heterologous desensitization of both CAP and MO responses was calcium-dependent. Further studies evaluating the role of calcineurin demonstrated that heterologous desensitization of CAP responses was calcineurin-dependent while heterologous desensitization of MO responses was calcineurin-independent. Homologous and heterologous desensitization of CAP and MO was also demonstrated using in vivo behavioral nocifensive assays. Taken together, these results indicate that TRPV1 and TRPA1 could be involved in a functional interaction that is regulated via different cellular pathways. The heterologous desensitization of these receptors and corresponding inhibition of nociceptor activity might have potential application as a therapeutic target for developing novel analgesics.