N-oleoyldopamine (OLDA), a bioactive lipid originally found in the mammalian brain, is an endovanilloids that selectively activates the transient receptor potential vanilloid type 1 (TRPV1) channel. This study tests the hypothesis that OLDA protects the heart against ischemia and reperfusion (I/R) injury via activation of the TRPV1 in wild type (WT) but not in gene-targeted TRPV1-null mutant (TRPV1^-/-) mice. Hearts of WT or TRPV1^-/- mice were Langendorffly perfused with OLDA (2 x 10^-9 M) in the presence or absence of CGRP8-37 (1 x 10^-6 M), a selective calcitonin gene-related peptide (CGRP) receptor antagonist; RP67580 (1 x 10^-6 M), a selective neurokinin-1 (NK1) receptor antagonist; chelerythrine (5 x 10^-6 M), a selective PKC antagonist; or tetrabutylammonium (TBA, 5 x 10^-4 M), a non selective K+ channel antagonist, followed by 35 minutes of global ischemia and 40 minutes of reperfusion (I/R). Left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), coronary flow (CF) and LV peak positive dP/dt (+dP/dt) were evaluated after I/R. OLDA improved recovery of cardiac function after I/R in WT but not TRPV1^-/- hearts by increasing LVDP, CF and +dP/dt, and by decreasing LVEDP. CGRP8-37, RP67580, chelerythrine, or TBA abolished the protective effect of OLDA in WT hearts. Radioimmunoassay showed that the release of SP and CGRP after OLDA treatment was higher in WT than in TRPV1^-/- hearts, which was blocked by chelerythrine or TBA. Thus, OLDA exerts a cardiac protective effect during I/R injury in WT hearts via CGRP and SP release, which is abolished by PKC- or K⁺ channel antagonists. The protective effect of OLDA is void in TRPV1^-/- hearts, supporting the notion that TRPV1 mediates OLDA induced protection against cardiac I/R injury.