The antifungal antibiotic clotrimazole (CLT) shows therapeutic effects on cancer, sickle cell disease, malaria, etc. by inhibiting membrane intermediate-conductance Ca2+ -activated K+ channels (IKCa). However, it is unclear whether this drug would affect human cardiac K+ currents. The present study was therefore designed to investigate the effects of CLT on transient outward K+ current (Ito1), and ultra-rapid delayed rectifier K+ current (IKur) in isolated human atrial myocytes, and cloned hERG channel current (IhERG) and recombinant human cardiac KCNQ1/KCNE1 channel current (IKs) expressed in HEK 293 cells. It was found that CLT inhibited Ito1 with an IC50 of 29.5 microM, accelerated Ito1 inactivation, and decreased recovery of Ito1 from inactivation. In addition, CLT inhibited human atrial I(Kur) in a concentration-dependent manner (IC50 = 7.6 microM). CLT substantially suppressed IhERG (IC50 = 3.6 microM), and negatively shifted the activation conductance of IhERG. Moreover, CLT inhibited IKs (IC50 = 15.1 microM), and positively shifted the activation conductance of the current. These results indicate that the antifungal antibiotic CLT substantially inhibits human cardiac repolarization K+ currents including Ito1, IKur, IhERG, and IKs. However, caution is recommended when correlating the observed in vitro effects on cardiac ion currents to the clinical relevance.