Volatile anesthetics protect the heart from ischemia/reperfusion injury but the mechanisms for this protection are poorly understood. Caveolae, sarcolemmal invaginations, and caveolins, scaffolding proteins in caveolae, localize molecules involved in cardiac protection. We tested the hypothesis that caveolae and caveolins are essential for volatile anesthetic-induced cardiac protection using cardiac myocytes (CMs) from adult rats and in vivo studies in caveolin-3 knockout mice (Cav-3^−/−). We incubated CM with methyl-β-cyclodextrin (MβCD) or colchicine to disrupt caveolae formation, and then exposed the myocytes to the volatile anesthetic isoflurane (30 min, 1.4%), followed by simulated ischemia/reperfusion (SI/R). Isoflurane protected CM from SI/R [23.2±1.6% vs. 71.0±5.8% cell death (assessed by trypan blue exclusion), P<0.001] but this protection was abolished by MβCD or colchicine (84.9±5.5% and 64.5±6.1% cell death, P<0.001). Membrane fractionation by sucrose density gradient centrifugation of CM treated with MβCD or colchicine revealed that buoyant (caveolae-enriched) fractions had decreased phosphocaveolin-1 and caveolin-3 compared to control CM. Cardiac protection in vivo was assessed by measurement of infarct size relative to the area at risk and cardiac troponin levels. Isoflurane-induced a reduction in infarct size and cardiac troponin relative to control (infarct size: 26.5%±2.6% vs. 45.3%±5.4%, P<0.01; troponin: 27.7±4.4 vs. 77.7±11.8 ng/ml, P<0.05). Isoflurane-induced cardiac protection was abolished in Cav-3^−/− mice (infarct size: 53.4%±6.1% vs. 53.2%±3.5%, P<0.01; troponin: 102.1±22.3 vs. 105.9±8.2 ng/ml, P<0.01). Isoflurane-induced cardiac protection is thus dependent on the presence of caveolae and the expression of caveolin-3. We conclude that caveolae and caveolin-3 are critical for volatile anesthetic-induced protection of the heart from ischemia/reperfusion injury.