The peripheral serotonergic system has been implicated in the modulation of an array of pain states, from migraine to fibromyalgia, however the mechanism by which serotonin (5HT) induces pain is unclear. Peripherally released 5HT induces thermal hyperalgesia, possibly via modulation of the transient receptor potential V1 (TRPV1) channel, which is gated by various noxious stimuli, including capsaicin. We previously reported in vitro that 5HT increases calcium accumulation in the capsaicin-sensitive population of sensory neurons with a corresponding increase in proinflammatory neuropeptide release, and both are antagonized by pretreatment with 5HT2A and 5HT3 antagonists, as well as the anti- migraine drug sumatriptan. In the current study, we extended these findings in vivo using the rat hindpaw thermal assay to test the hypothesis that peripheral 5HT enhances TRPV1- evoked thermal hyperalgesia that can be attenuated with 5HT2A and 5HT3 receptor antagonists, as well as sumatriptan. Thermal hyperalgesia and edema were established by 5HT injection (0.1-10 nmol/100 μL) into the rat hindpaw and the latency to paw withdrawal (PWL) from noxious heat was determined. Rats were then pretreated with either 5HT prior to capsaicin (3 nmol/10 μl), the 5HT2A receptor antagonist ketanserin or the 5HT3 receptor antagonist granisetron (0.0001-0.1 nmol/100 μL) prior to 5HT and/or capsaicin, or the 5HT1B/1D receptor agonist sumatriptan (0.01-1 nmol/100 μL) prior to capsaicin and PWL was determined. We report that 5HT pretreatment enhances TRPV1- evoked thermal hyperalgesia, which is attenuated with local pretreatment with ketanserin, granisetron or sumatriptan. We also report that peripheral 5HT induced a similar magnitude of thermal hyperalgesia in male and female rats. Overall, our results provide in vivo evidence supporting an enhancing role of 5HT on TRPV1-evoked thermal hyperalgesia, which can be attenuated by peripheral serotonergic intervention.