Updated project metadata. Dilated cardiomyopathy (DCM) is a major risk factor for developing heart failure and is often associated with an increased risk for life-threatening arrhythmia. Although numerous causal genes for DCM have been identified, RNA binding motif 20 (RBM20) remains one of the few splicing factors that, when mutated or genetically ablated, leads to the development of DCM. In this study we sought to identify changes in the cardiac proteome in RBM20 deficient rat hearts using global quantitative proteomics to gain insight into the molecular mechanisms precipitating the development of DCM secondary to RBM20 loss. Our analysis identified changes in titin interacting proteins, as well as mitochondrial enzymes, implicating activation of pathological hypertrophy and mitochondrial dysfunction in DCM development in RBM20 deficient rats. Collectively, our findings provide the first look into changes in the cardiac proteome associated with genetic ablation of RBM20.