• Protein kinase C differentially regulates Kir6.2 and SUR2A expression in early vs. late exercise preconditioning.
• Inhibition of protein kinase C alters mRNA and protein levels of SarcKATP channel subunits in a phase-specific manner.
• The regulatory effects of protein kinase C on Kir6.2 and SUR2A are coordinated and complementary across early and late preconditioning.
• These findings provide insights into the molecular mechanisms underlying exercise-induced cardioprotection.