MicroRNAs are small endogenous noncoding RNAs that regulate protein expression by hybridization to imprecise complementary sequences of target mRNAs. Changes in abundance of muscle-specific microRNA, miR-1, have been implicated in cardiac disease, including arrhythmia and heart failure. However, the specific molecular targets and cellular mechanisms involved in the action of miR-1 in the heart are only beginning to emerge. In this study we investigated the effects of increased expression of miR-1 on excitation–contraction coupling and Ca²⁺ cycling in rat ventricular myocytes using methods of electrophysiology, Ca²⁺ imaging and quantitative immunoblotting. Adenoviral-mediated overexpression of miR-1 in myocytes resulted in a marked increase in the amplitude of the inward Ca²⁺ current, flattening of Ca²⁺ transients voltage dependence, and enhanced frequency of spontaneous Ca²⁺ sparks while reducing the sarcoplasmic reticulum Ca²⁺ content as compared with control. In the presence of isoproterenol, rhythmically paced, miR-1–overexpressing myocytes exhibited spontaneous arrhythmogenic oscillations of intracellular Ca²⁺, events that occurred rarely in control myocytes under the same conditions. The effects of miR-1 were completely reversed by the CaMKII inhibitor KN93. Although phosphorylation of phospholamban was not altered, miR-1 overexpression increased phosphorylation of the ryanodine receptor (RyR2) at S2814 (Ca²⁺/calmodulin-dependent protein kinase) but not at S2808 (protein kinase A). Overexpression of miR-1 was accompanied by a selective decrease in expression of the protein phosphatase PP2A regulatory subunit B56α involved in PP2A targeting to specialized subcellular domains. We conclude that miR-1 enhances cardiac excitation–contraction coupling by selectively increasing phosphorylation of the L-type and RyR2 channels via disrupting localization of PP2A activity to these channels.