It remains to be elucidated whether Ca antagonists induce pharmacological preconditioning to protect the heart against ischemia/reperfusion injury. The aim of this study was to determine whether and how pretreatment with a Ca antagonist, azelnidipine, could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury in vitro. Using HL-1 cardiomyocytes, we studied effects of azelnidipine on NO synthase (NOS) expression, NO production, cell death and apoptosis during H/R. Action potential durations (APDs) were determined by the whole-cell patch-clamp technique. Azelnidipine enhanced endothelial NOS phosphorylation and NO production in HL-1 cells under normoxia, which was abolished by a heat shock protein 90 inhibitor, geldanamycin, and an antioxidant, N-acetylcysteine. Pretreatment with azelnidipine reduced cell death and shortened APDs during H/R. These effects of azelnidipine were diminished by a NOS inhibitor, L-NAME, but were influenced by neither a T-type Ca channel inhibitor, NiCl, nor a N-type Ca channel inhibitor, ω-conotoxin. The azelnidipine-induced reduction in cell death was not significantly enhanced by either additional azelnidipine treatment during H/R or increasing extracellular Ca concentrations. RNA sequence (RNA-seq) data indicated that azelnidipine-induced attenuation of cell death, which depended on enhanced NO production, did not involve any significant modifications of gene expression responsible for the NO/cGMP/PKG pathway. We conclude that pretreatment with azelnidipine protects HL-1 cardiomyocytes against H/R injury via NO-dependent APD shortening and L-type Ca channel blockade independently of effects on gene expression.© 2024. Springer Nature Japan KK, part of Springer Nature.