Transient receptor potential vanilloid subtype 1 (TRPV1) is known as capsaicin (CAP) receptor and activated by CAP. Activation of TRPV1 by CAP increases energy expenditure and thermogenesis in rodents or human. Therefore, TRPV1 may be target for energy expenditure enhancement and thermogenesis. To search for novel TRPV1 agonist, we screened 19 types of foods by using TRPV1-expressing HEK293 cells. TRPV1 was activated by hexane extract of wheat flour, and its functional compounds were 1-monoacylglycerols containing oleic, linoleic, and alpha-linolenic acids. Their potencies (EC50) were about 50 times larger than that of CAP and their efficacies (maximal response) were about half of that of CAP. TRPV1 was activated by 1-monoacylglycerols (MGs) having C18 and C20 unsaturated and C8-C12 saturated fatty acid (FA). Moreover, 2-MGs having C18 and C20 unsaturated FA acted on TRPV1 with the same potency. On the other hand, no activation of TRPV1 was induced by MGs having C16 and C18 saturated FA, di- or triacylglycerols of C18:1 FA. Pain-relating aversive responses were induced when TRPV1-activating 1-monoacylglycerols (50 mM) was administered subcutaneously into rat hind paw. These effects were inhibited by the co-injection of capsazepine (10 mM) which is a TRPV1 competitive antagonist. These results suggested that these 1-monoacylglycerols activate TRPV1 in vitro and in vivo.