A variety of lipid and lipid-derived molecules can modulate TRP cation channel activity, but the identity of the lipids that affect TRP channel function in vivo is unknown. Here, we use genetic and behavioral analysis in the nematode C. elegans to implicate a subset of 20-carbon polyunsaturated fatty acids (PUFAs) in TRPV channel-dependent olfactory and nociceptive behaviors. Olfactory and nociceptive TRPV signaling are sustained by overlapping but nonidentical sets of 20-carbon PUFAs including eicosapentaenoic acid (EPA) and arachidonic acid (AA). PUFAs act upstream of TRPV family channels in sensory transduction. Short-term dietary supplementation with PUFAs can rescue PUFA biosynthetic mutants, and exogenous PUFAs elicit rapid TRPV-dependent calcium transients in sensory neurons, bypassing the normal requirement for PUFA synthesis. These results suggest that a subset of PUFAs with omega-3 and omega-6 acyl groups act as endogenous modulators of TRPV signal transduction.