Recent studies have demonstrated that intralipid (ILP) conferred myocardial protection against ischemia-reperfusion (IR) injury through activation of reperfusion injury salvage kinase (RISK) pathway. As RISK signal has been shown to be impaired in hypertrophied myocardium, we investigated whether ILP-induced cardiac protection was maintained in hypertrophied rat hearts. Transverse aortic constriction was performed on male Sprague-Dawley rats to induce left ventricular hypertrophy, then sham-operated or hypertrophied rat hearts were isolated and perfused retrogradely by the Langendorff for 30 min (equilibration) followed by 40 min of ischemia and then 120 min of reperfusion. The isolated hearts received 15-min episode of 1% ILP separated by 15 min of washout or three episodes of 5-min ischemia followed by 5-min reperfusion before ischemia. The hemodynamics, infarct size, apoptosis, phosphorylated protein kinase B (p-Akt), phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2), phosphorylated glycogen synthase kinase 3β (GSK3β), Bcl-2, phosphorylated Bad, and Bax were determined. We found that ILP significantly improved left ventricular hemodynamics and reduced infarct size and the number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)–positive cells in the sham-operated rat hearts exposed to IR. However, such myocardial infarct–sparing effect of ILP was completely blocked by phosphatidylinositol-3-kinase inhibitor wortmannin, but only partially by mitogen-activated protein kinase kinase inhibitor PD98059 in sham-operated hearts. Intralipd upregulated the phosphorylation of Akt, extracellular regulated protein kinase 1/2 (ERK1/2), and their downstream target of GSK3β and antiapoptotic Bcl-2 expression in healthy rat hearts. Nonetheless, ILP failed to improve left ventricular hemodynamics and reduced infarct size and apoptosis and increase the phosphorylated Akt, ERK1/2, GSK3β, and antiapoptotic Bcl-2 in hypertrophied myocardium. In contrast, ischemic preconditioning increased the phosphorylation of Akt, ERK1/2 and GSK3β, improved heart pump function, and reduced myocardial necrosis in sham-operated hearts, a phenomenon partially attenuated by ventricular hypertrophy. Interestingly, GSK inhibitor SB216763 conferred cardioprotection against IR injury in sham-operated hearts, but failed to exert cardioprotection in hypertrophied myocardium. Our results indicated that ventricular hypertrophy abrogated ILP-induced cardioprotection against IR injury by alteration of RISK/GSK3β signal.