MuRF1 is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine if MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Tibialis anterior (TA) muscles were transfected with an untagged MuRF1 plasmid or control plasmid for 14 days. A total of 963 ubiquitination sites, corresponding to 250 proteins, were quantified from the TA muscle. Statistical analysis revealed that the overexpression of MuRF1 resulted in significant upregulation of 153 ubiquitination sites on 45 proteins and significant downregulation of 16 sites on 11 proteins. Substrates of MuRF1 include contractile and metabolic proteins, deubiquitinases, p62, and VCP. Moreover, MuRF1-mediated ubiquitination leads to destabilization and breakdown of the sarcomere and reveals a role for MuRF1 in the regulation of additional proteolytic pathways in skeletal muscle.