We have previously shown that ischemic preconditioning (IPC) protection against necrosis in whole hearts and both fresh and cultured cardiomyocytes, as well as improved regulatory volume decrease (RVD) to hypo-osmotic swelling in cardiomyocytes, are abrogated through Cl- channel blockade, pointing to a role for enhanced cell volume regulation in IPC. To further define this cardioprotective mechanism, cultured rabbit ventricular cardiomyocytes were preconditioned either by 10-minute simulated ischemia (SI) followed by 10-minute simulated reperfusion (SR), by 10-minute exposure/10-minute washout of remote ischemic preconditioning (rIPC) plasma dialysate (from rabbits subjected to repetitive limb ischemia), or by adenoviral transfection with the constitutively active PKC gene. These interventions were done prior to subjecting the cardiomyocytes to either 60-minute or 75-minute SI/60-minute SR to assess cell necrosis (by trypan blue staining), 30-minute SI to assess ischemic cell swelling, or 30-minute hypo-osmotic (200 mOsm) stress (HS) to assess cell volume regulation. Necrosis after SI/SR and both the SI- and HS-induced swelling was reduced in preconditioned cardiomyocytes compared to control cardiomyocytes (neither preconditioned nor transfected). These effects on necrosis and cell swelling were blocked by either Cl^- channel blockade or dominant-negative knockdown of IK1 channels with adenoviruses, suggesting that Cl^- and K⁺ movement across the sarcolemma are critical for cell volume regulation and thereby cell survival under hypoxic/ischemic conditions. Our results define enhanced cell volume regulation as a key common mechanism of cardioprotection by preconditioning in cardiomyocytes.