Background: Dysregulation of protein kinase–mediated signaling is an early event in many diseases, including the most common clinical cardiac arrhythmia, atrial fibrillation (AF). Kinomic profiling represents a promising technique to identify candidate kinases. Objective: In this study we used kinomic profiling to identify kinases altered in AF remodeling using atrial tissue from a canine model of AF (atrial tachypacing). Methods: Left atrial tissue obtained in a previous canine study was used for kinomic array (containing 1024 kinase pseudosubstrates) analysis. Three groups of dogs were included: nonpaced controls and atrial tachypaced dogs, which were contrasted with geranylgeranylacetone-treated dogs with AF, which are protected from AF promotion, to enhance specificity of detection of putative kinases. Results: While tachypacing changed activity of 50 kinases, 40 of these were prevented by geranylgeranylacetone and involved in differentiation and proliferation (SRC), contraction, metabolism, immunity, development, cell cycle (CDK4), and survival (Akt). Inhibitors of Akt (MK2206) and CDK4 (PD0332991) and overexpression of a dominant-negative CDK4 phosphorylation mutant protected against tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes. Moreover, patients with AF show down- and upregulation of SRC and Akt phosphorylation, respectively, similar to findings of the kinome array. Conclusion: Contrasting kinomic array analyses of controls and treated subjects offer a versatile tool to identify kinases altered in atrial remodeling owing to tachypacing, which include Akt, CDK4, and SRC. Ultimately, pharmacological targeting of altered kinases may offer novel therapeutic possibilities to treat clinical AF.