The receptor channel transient receptor potential vanilloid 1 (TRPV1) functions as a sensor of noxious heat and various chemicals. There is increasing evidence for a crosstalk between TRPV1 and opioid receptors. Here we investigated the effect of the prototypical TRPV1 agonist capsaicin and selected opioid ligands on TRPV1 movement in the plasma membrane and intracellular calcium levels in HEK293 cells expressing TRPV1 tagged with cyan fluorescent protein (CFP). We observed that lateral mobility of TRPV1 increased after treatment of cells with capsaicin or naloxone (a nonselective opioid receptor antagonist) but not with DAMGO (a μ-opioid receptor agonist). Interestingly, both capsaicin and naloxone, unlike DAMGO, elicited intracellular calcium responses. The increased TRPV1 movement and calcium influx induced by capsaicin and naloxone were blocked by the TRPV1 antagonist capsazepine. The ability of naloxone to directly interact with TRPV1 was further corroborated by 3H-naloxone binding. In conclusion, our data suggest that besides acting as an opioid receptor antagonist, naloxone may function as a potential TRPV1 agonist.