Cardiomyocytes exposed to ischemia followed by reperfusion undergo apoptosis, some of which is induced via the mitochondrial pathway, and some of which is induced via the death-receptor ligand pathway. FLICE-inhibitory protein (FLIP) is a cellular protein that, in overexpression experiments in other cell types, has been shown to be capable of either inducing or protecting from death-receptor-mediated apoptosis, depending on the extent of overexpression. To examine the role of endogenous FLIP in neonatal cardiomyocytes we have used short hairpin-induced (shRNA) silencing generated from a transfected plasmid. Silencing of FLIP increases the background level of apoptosis detected by TUNEL assay in primary cardiomyocytes and sensitizes the cells to apoptosis induced by simulated ischemia/reoxygenation (IR). This result indicates that previous observations of decreased FLIP levels and increased apoptosis in cardiac infarcts is more than just correlative, but reflects an innate protective role of FLIP in cardiomyocytes. We also found that mitochondrial activity, as determined by MitoTracker red staining, is reduced in the absence of FLIP, and further decreases after exposure to simulated IR. Conversely, overexpression of FLIP somewhat prevents the decrease in mitochondrial activity after simulated IR. Thus, FLIP may confer protection from simulated ischemia/reperfusion through more than one pathway.