Although the transient receptor potential vanilloid type 1 (TRPV1) -containing afferent nerve fibers are widely distributed in the heart, the relationship between TRPV1 function and cardiac ischemic preconditioning (PC) has not been well defined. Using TRPV1 knockout mice, we studied the role of TRPV1 in PC-induced myocardial protection. Hearts of gene targeted TRPV1-null mutant (TRPV1 ^-/-) or wild-type (WT) mice were Langendorffly perfused in the presence or absence of CGRP8-37, a selective calcitonin gene-related peptide (CGRP) receptor antagonist; or RP67580, a selective neurokinin 1 (NK1) receptor antagonist when hearts were subjected to three 5-mins of ischemia PC followed by 30-mins of global ischemia and 40 minutes of reperfusion (I/R). PC prior to I/R decreased left ventricular end-diastolic pressure (LVEDP) and increased left ventricular developed pressure (LVDP), coronary flow (CF), LV peak positive dP/dt (+dP/dt) in WT mice (PC-WT) compared to PC-TRPV1 ^-/-, TRPV1 ^-/-, or WT hearts (p<0.05), and PC also decreased LVEDP in PC-TRPV1 ^-/- compared to TRPV1 ^-/-. CGRP8-37 or RP67580 abolished PC induced protection in WT but not TRPV1 ^-/- hearts (p<0.05). Moreover, PC decreased lactate dehydrogenase (LDH) release and infarct size in PC-WT compared to PC-TRPV1 ^-/-, TRPV1 ^-/-, or WT hearts, and it also lowered these parameters in PC-TRPV1 ^-/- compared to TRPV1 ^-/- hearts (p<0.05). Radioimmunoassay showed that the release of substance P (SP) and CGRP after PC was higher in WT hearts than in TRPV1 ^-/- hearts (p<0.05), which was attenuated by CAPZ in WT but not TRPV1 ^-/- hearts. Thus, PC induced protection of the heart was impaired in TRPV1 ^-/- hearts, indicating that TRPV1 contributes to the beneficial effects of preconditioning against I/R injury through release SP and CGRP.