The multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is activated by elevated intracellular Ca²⁺ (Ca²⁺_i), and mice with chronic myocardial CaMKII inhibition (Inh) resulting from transgenic expression of a CaMKII inhibitory peptide (AC3-I) unexpectedly showed action potential duration (APD) shortening. Inh mice exhibit increased L-type Ca²⁺ current (I_Ca), because of upregulation of protein kinase A (PKA) activity, and decreased CaMKII-dependent phosphorylation of phospholamban (PLN). We hypothesized that CaMKII is a molecular signal linking Ca²⁺_i to repolarization. Whole cell voltage-clamp recordings revealed that the fast transient outward current (I_to,f) and the inward rectifier current (I_K1) were selectively upregulated in Inh, compared with wild-type (WT) and transgenic control, mice. Breeding Inh mice with mice lacking PLN returned I_to,f and I_K1 to control levels and equalized the APD and QT intervals in Inh mice to control and WT levels. Dialysis of AC3-I into WT cells did not result in increased I_to,f or I_K1, suggesting that enhanced cardiac repolarization in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. Increasing PKA activity, by cell dialysis with cAMP, or inhibition of PKA did not affect I_K1 in WT cells. Dialysis of WT cells with cAMP also reduced I_to,f, suggesting that PKA upregulation does not increase repolarizing K⁺ currents in Inh mice. These findings provide novel in vivo and cellular evidence that CaMKII links Ca²⁺_i to cardiac repolarization and suggest that PLN may be a critical CaMKII target for feedback regulation of APD in ventricular myocytes.