Objective To observe the changes of transient outward potassium currents (I_to) in single ventricular myocytes after establishment of an acute hypoxic exercise model in rats, and explore the effects of exhaustive exercise on cardiac electrophysiology under simulated altitude hypoxia conditions at the cellular level. Methods Forty healthy male clean SD rats were randomly divided into hypoxic exercise group, hypoxic resting group, normoxic exercise group and normoxic resting group, with 10 rats in each group. Small animal hypobaric oxygen chamber was used to simulate the hypoxia or normal oxygen condition for exhaustive exercise test. The heart of each group of rats was taken out, and the single rat ventricular myocytes were separated by perfusion enzymatic hydrolysis. The whole cell patch clamp technique was used to record I_to current of the rat single ventricular myocytes. SPSS statistics 20.0 was used for data processing, ANOVA analysis of variance was applied for comparison among multiple groups, and SNK-q test was adopted for pairwise comparison between groups. Results Compared with the normoxic resting group, at +40 mV, the I_to current density of the hypoxic exercise group was significantly lower, and was lower than that of the hypoxic resting group and normoxia exercise group. This effect was in a voltage-dependent manner. Studies on the gating mechanism showed that the I_to steady-state activation curve of rat cardiomyocytes shifted to the hyperpolarization direction during hypoxic exercise, while the steady-state inactivation curve shifted to the depolarization direction. The combination of above 2 effects made the I_to current significantly reduced significantly. Conclusion Acute hypoxic exercise can reduce the I_to current of rat ventricular myocytes by changing the steady-state activation and steady-state inactivation of potassium channels, which may be one of the main reasons for acute hypoxic exercise to cause arrhythmia.