Dynamin-2 is a ubiquitously expressed GTP-ase that mediates membrane remodeling. Recent findings indicate that dynamin-2 also regulates actin dynamics. Mutations in dynamin-2 cause dominant centronuclear myopathy (CNM), a congenital myopathy characterized by progressive weakness and atrophy of skeletal muscles. However, the muscle-specific roles of dynamin-2 affected by these mutations remain elusive. Here we show that, in muscle cells, the GTP-ase activity of dynamin-2 is involved in de novo actin polymerization as well as in actin-mediated trafficking of the glucose transporter GLUT4. Expression of dynamin-2 constructs carrying CNM-linked mutations disrupted the formation of new actin filaments as well as the stimulus-induced translocation of GLUT4 to the plasma membrane. Similarly, mature muscle fibers isolated from heterozygous knock-in mice that harbor the dynamin-2 mutation p.R465W, an animal model of CNM, exhibited altered actin organization, reduced actin polymerization and impaired insulin-induced translocation of GLUT4 to the sarcolemma. Moreover, GLUT4 displayed aberrant perinuclear accumulation in biopsies from CNM patients carrying dynamin-2 mutations, further suggesting trafficking defects. These results suggest that dynamin-2 is a key regulator of actin dynamics and GLUT4 trafficking in muscle cells. Our findings also support a model in which impairment of actin-dependent trafficking contributes to the pathological mechanism in dynamin-2-associated CNM. Dynamins are mechano-chemical large GTP-ases, whose catalytic activity is required in several membrane-based processes including endocytosis, vesicle trafficking, and exocytosis 1-4. These proteins also exhibit a critical role in actin cytoskeleton dynamics by promoting elongation 5 , remodeling 6 and stabilizing actin filaments 7. Dynamins are composed of five conserved domains: an N-terminal GTP-ase domain, a middle structural domain, a pleckstrin homology (PH) domain that binds phosphoinositides, a GTP-ase effector domain (GED), and a C-terminal proline/arginine-rich domain (PRD) that binds SH3-domain-containing partners 1-4. Three dynamin isoforms have been described in mammals, which share approximately 80% of sequence homology