Mesenchymal stem cell (MSC) infusion may reduce myocardial ischemic injury. TNF-alpha is a proinflammatory cytokine produced in large quantities during myocardial ischemia that can exert beneficial or detrimental effects on MSC function by binding to a 55-kd receptor (TNFR1) or a 75-kd receptor (TNFR2) on MSCs. We investigated whether genetic modification with ablation of TNFR1 and/or TNFR2 affects MSC-mediated protection against myocardial ischemic injury. The MSCs were harvested from wild-type mice (WT-MSCs) and knockout mice with ablation of TNFR1 and/or TNFR2 (TNFR1KO, TNFR2KO, and TNFR1/R2KO MSCs). After anesthesia was initiated via inhalation of isoflurane, myocardial ischemia was induced in rats via coronary artery ligation. Hearts were then injected with vehicle or MSCs (1 x 10 cells/mL). Myocardial function was assessed 28 days postsurgery with 2-dimensional echocardiograms and isolated heart perfusion. Myocardial tissue was collected for cytokine analysis and infarct measurements. We found that MSC treatment offered significant protection against myocardial ischemia, namely by decreasing infarct size, improving heart function, and decreasing ventricular remodeling compared with vehicle. Compared with WT-MSCs, TNFR1KO MSCs conferred increased cardiac protection, although TNFR2KO and TNFR1/R2KO MSCs conferred less cardiac protection. In addition, treatment with TNFR1KO MSCs was associated with decreased levels of proinflammatory cytokines and an increased level of vascular endothelial growth factor in the myocardium, whereas treatment with TNFR2KO or TNFR1/R2KO MSCs was associated with increased levels of proinflammatory cytokines and a decreased level of vascular endothelial growth factor compared with treatment with WT-MSCs. We conclude that MSC TNFR1 and TNFR2 play important roles in MSC-mediated cardiac protection after myocardial ischemia.