The human ether-a-go-go-related gene (hERG) inhibition is a serious cardiac safety issue. Although the inhibition of hERG by majority of compounds are stable, the strength of the inhibition of a few compounds, including Erythromycin, is remarkably rising from room temperature (RT) to physiological temperature (PT). Understanding the features of erythromycin against hERG could help us to decide which compounds are needed for further study. The whole cell patch clamp technique was used to investigate the effects of erythromycin on hERG channels in different temperatures. We found that erythromycin caused a concentration dependent inhibition of cardiac hERG potassium channels. The half maximal inhibitory concentration (IC50) value was 1671 ± 593 μM (n = 5-10) at RT, about 11 folds higher than its IC50 (150 ± 26 μM, n = 7) at PT. Although temperature does have a profound change of hERG channel dynamic, the erythromycin further left shifted channel’s steady state activation, steady state inactivation, and make onset of inactivation significantly faster at both temperatures. It is interesting that our data suggests that there is critical binding site shifted from V625 at RT to Y652 at PT. By contrast, Cisapride, a well-known hERG blocker and its inhibition is not affected by temperature, does not change its critical binding sites after the temperature is raised to PT. The data suggests that increase strength of the inhibition could be duo to the shift of its binding sites of hERG channels.