Pioglitazone (PIO), a PPAR-gamma agonist, limits myocardial infarct size by activating Akt and upregulating cytosolic phospholipase A(2) (cPLA(2)) and cyclooxygenase (COX)-2. However, PIO has several PPAR-gamma-independent effects. We assessed whether PIO limits myocardial infarct size in PPAR-gamma-knockout mice, attenuates hypoxia-reoxygenation injury and upregulates P-Akt, cPLA(2), and COX-2 expression in PPAR-gamma-knockout cardiomyocytes. Cardiac-specific inducible PPAR-gamma knockout mice were generated by crossing alpha MHC-Cre mice to PPAR-gamma(loxp/loxp) mice. PPAR-gamma deletion was achieved after 7 days of intraperitoneal tamoxifen (20 mg/kg/day) administration. Mice received PIO (10 mg/kg/day), or vehicle, for 3 days and underwent coronary occlusion (30 min) followed by reperfusion (4 h). We assessed the area at risk by blue dye and infarct size by TTC. Cultured adult cardiomyocytes of PPAR-gamma(loxp/loxp/cre) mice without or with pretreatment with tamoxifen were incubated with or without PIO and subjected to 2 h hypoxia/2 h reoxygenation. Cardiac-specific PPAR-gamma knockout significantly increased infarct size. PIO reduced infarct size by 51% in PPAR-gamma knockout mice and by 55% in mice with intact PPAR-gamma. Deleting the PPAR-gamma gene increased cell death in vitro. PIO reduced cell death in cells with and without intact PPAR-gamma. PIO similarly increased myocardial Ser-473 P-Akt, cPLA(2), and COX-2 levels after hypoxia/reoxygenation in cells with and without intact PPAR-gamma. PIO limited infarct size in mice in a PPAR-gamma-independent manner. PIO activated Akt, increased the expression of cPLA(2) and COX-2, and protected adult cardiomyocytes against the effects of hypoxia/reoxygenation independent of PPAR-gamma activation.