To sustain cardiac muscle contractility relatively independent of temperature, some fish species are capable of temporarily altering excitation-contraction (E-C) coupling processes to meet the demands of their environment. The Pacific bluefin tuna, Thunnus orientalis, is a partially endothermic fish that inhabits a wide range of thermal niches. The present study examined the effects of temperature and thermal acclimation on sarcolemmal K⁺ currents and their role in action potential (AP) generation in bluefin tuna cardiomyocytes. Atrial and ventricular myocytes were enzymatically isolated from cold- (14^oC) and warm- (24^oC) acclimated bluefin tuna. AP's and current-voltage relations of K⁺ channels were measured using the whole-cell current and voltage clamp techniques, respectively. Data were collected either at the cardiomyocytes' respective acclimation temperature of 14 or 24°C or at a common test temperature of 19°C (to reveal the effects of acclimation). AP duration (APD) was prolonged in cold-acclimated (CA) cardiomyocytes tested at 14^oC compared to 19°C and in warm-acclimated (WA) cardiomyocytes tested at 19°C compared with 24°C. This effect was mirrored by a decrease in the density of the delayed rectifier current (I_Kr), while the density of the background inward rectifier current (I_K1) was unchanged. When CA and WA cardiomyocytes were tested at a common temperature of 19°C, no significant effects of temperature acclimation on AP shape or duration were observed, while I_Kr density was markedly increased in CA cardiomyocytes. I_K1 density was unaffected in CA ventricular myocytes but was significantly reduced in CA atrial myocytes, resulting in a depolarization of atrial resting membrane potential (Vm). Our results indicate the bluefin AP is relatively short compared to other teleosts, which may allow the bluefin heart to function at cold temperatures without the necessity for thermal compensation of APD.