Capsaicin, an agonist of TRPV1, evokes intracellular [Ca2+] transients and glutamate release from perfused trigeminal ganglion. The spider toxin PnTx3-5, native or recombinant is more potent than the selective TRPV1 blocker SB-366791 with IC50 of 47?±?0.18?nM, 45?±?1.18?nM and 390?±?5.1?nM in the same experimental conditions. PnTx3-5 is thus more potent than the selective TRPV1 blocker SB-366791. PnTx3-5 (40?nM) and SB-366791 (3?μM) also inhibited the capsaicin-induced increase in intracellular Ca2+ in HEK293?cells transfected with TRPV1 by 75?±?16% and 84?±?3.2%, respectively. In HEK293?cells transfected with TRPA1, cinnamaldehyde (30?μM) generated an increase in intracellular Ca2+ that was blocked by the TRPA1 antagonist HC-030031 (10?μM, 89% inhibition), but not by PnTx3-5 (40?nM), indicating selectivity of the toxin for TRPV1. In whole-cell patch-clamp experiments on HEK293?cells transfected with TRPV1, capsaicin (10?μM) generated inward currents that were blocked by SB-366791 and by both native and recombinant PnTx3-5 by 47?±?1.4%; 54?±?7.8% and 56?±?9.0%, respectively. Intradermal injection of capsaicin into the rat left vibrissa induced nociceptive behavior that was blocked by pre-injection with either SB-366791 (3 nmol/site i.d., 83.3?±?7.2% inhibition) or PnTx3-5 (100 fmol/site, 89?±?8.4% inhibition). We conclude that both native and recombinant PnTx3-5 are potent TRPV1 receptor antagonists with antinociceptive action on pain behavior evoked by capsaicin.,Copyright ? 2019. Published by Elsevier Ltd.