The Ca²⁺-independent phospholipase A2 group VI (PLA₂-VI) and the Na⁺/H⁺ exchanger isoform 1 (NHE1) are highly pH sensitive proteins which exert both protective and detrimental effects in cardiac ischemia/reperfusion. Here, we investigated the role of pHo in ischemia/reperfusion injury and death, and in regulation and function of iPLA₂-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI, 0.5% O2, 8 mM K⁺, 20 mM lactate) at pHo 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared to neutral SI, while necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA₂-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by 5'N-ethylisopropylamiloride (EIPA) had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA₂-VI disrupted F-actin, cortactin, and mitochondrial integrity, while inhibition of NHE slightly reduced stress fibre content. iPLA₂-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pHo dependent manner during SI/R, which also affected the subcellular localization of both proteins. Thus, the mode of cell death, and the roles and regulation of iPLA₂-VI and NHE1, are at least in part determined by the pH_o during SI. In addition to having clinically relevant implications, these finding can in part explain the contradictory results obtained from previous studies of iPLA₂-VIA and NHE1 during cardiac ischemia/reperfusion.