DOCK (dedicator of cytokinesis) proteins are 11-member family of typical guanine exchange factors (GEFs) expressed almost exclusively in the brain and sprain cord. We previously identified DOCK3 as being strongly upregulated in Duchenne muscular dystrophy (DMD) patient and mouse skeletal muscles DOCK3 is an essential modulator of PTEN/AKT pathway and has been shown to be essential for myoblast fusion and migration. Here, we aimed to characterize the function of DOCK3 in normal and dystrophin-deficient mice. Dock3 KO mice on the dystrophin-deficient background exacerbated skeletal muscle and cardiac phenotypes. Dock3 KO mice developed poor muscle architecture, locomotive activity, and impaired skeletal muscle regeneration. Additionally, DOCK3 KO mice had significant cardiac defects similar to those observed in DMD mouse models. Together, these studies demonstrate a functional role for DOCK3 in normal and DMD muscle function and a biomarker for DMD disease progression.