The rapidly activating delayed rectifier potassium current (IKr) plays a critical role in cardiac repolarization. Although IKr is known to be regulated by both α1- and β1-adrenergic receptors (ARs), the cross-talk and feedback mechanisms that dictate its response to α1- and β1-AR activation are not known. In the present study, IKr was recorded using the whole-cell patch-clamp technique. IKr amplitude was measured before and after the sequential application of selective adrenergic agonists targeting α1- and β1-ARs. Stimulation of either receptor alone (α1-ARs using 1 μM phenylephrine (PE) or β1-ARs using 10 μM xamoterol (Xamo)) reduced IKr by 0.22 ± 0.03 and 0.28 ± 0.01, respectively. The voltage-dependent activation curve of IKr shifted in the negative direction. The half-maximal activation voltage (V0.5) was altered by -6.35 ± 1.53 and -1.95 ± 2.22 mV, respectively, with no major change in the slope factor (k). When myocytes were pretreated with Xamo, PE-induced reduction in IKr was markedly blunted and the corresponding change in V0.5 was significantly altered. Similarly, when cells were pretreated with PE, Xamo-induced reduction of IKr was significantly attenuated. The present results demonstrate that functional cross-talk between α1- and β1-AR signaling regulates IKr. Such non-linear regulation may form a protective mechanism under excessive adrenergic stimulation.