Background The multifunctional Ca ²⁺ ‐ and calmodulin‐dependent protein kinase II (Ca MKII ) is a crucial mediator of cardiac physiology and pathology. Increased expression and activation of Ca MKII has been linked to elevated risk for arrhythmic events and is a hallmark of human heart failure. A useful approach to determining Ca MKII 's role therein is large‐scale analysis of phosphorylation events by mass spectrometry. However, current large‐scale phosphoproteomics approaches have proved inadequate for high‐fidelity identification of kinase‐specific roles. The purpose of this study was to develop a phosphoproteomics approach to specifically identify Ca MKII 's downstream effects in cardiac tissue. Methods and Results To identify putative downstream Ca MKII targets in cardiac tissue, animals with myocardial‐delimited expression of the specific peptide inhibitor of Ca MKII ( AC 3‐I) or an inactive control ( AC 3‐C) were compared using quantitative phosphoproteomics. The hearts were isolated after isoproterenol injection to induce Ca MKII activation downstream of β‐adrenergic receptor agonist stimulation. Enriched phosphopeptides from AC 3‐I and AC 3‐C mice were differentially quantified using stable isotope dimethyl labeling, strong cation exchange chromatography and high‐resolution LC ‐ MS / MS . Phosphorylation levels of several hundred sites could be profiled, including 39 phosphoproteins noticeably affected by AC 3‐I‐mediated Ca MKII inhibition. Conclusions Our data set included known Ca MKII substrates, as well as several new candidate proteins involved in functions not previously implicated in Ca MKII signaling.