To gain better understanding of the detailed mechanisms of high-intensity focused ultrasound (HIFU) ablation for cardiac arrhythmias, we investigated how the cellular electrophysiological (EP) changes were correlated with temperature increases and thermal dose (cumulative equivalent minutes [CEM43]) during HIFU application using Langendorff-perfused rabbit hearts. Employing voltage-sensitive dye di-4-ANEPPS, we measured the EP and temperature during HIFU using simultaneous optical mapping and infrared imaging. Both action potential amplitude (APA) and AP duration at 50% repolarization (APD50) decreased with temperature increases, and APD50 was more thermally sensitive than APA. EP and tissue changes were irreversible when HIFU-induced temperature increased above 52.3 ± 1.4 °C and log10(CEM43) above 2.16 ± 0.51 (n = 5), but were reversible when temperature was below 50.1 ± 0.8 °C and log10(CEM43) below −0.9 ± 0.3 (n = 9). EP and temperature/thermal dose changes were spatially correlated with HIFU induced tissue necrosis surrounded by a transition zone.