DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine–threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast–myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. ► Expression of DMPK in the cytosol induces formation of stellar actomyosin stress fibers. ► Stellar stress fiber formation is accompanied by augmented phosphorylation of MLC2. ► Myoblasts expressing active cytosolic DMPK display altered cell morphology and motility. ► Early steps in myogenesis are delayed upon DMPK activity expression in the cytosol. ► High cytosolic DMPK activity during myogenesis supports formation of myosacs.