Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, leads to progressive and fatal muscle weakness through yet-to-be-fully deciphered molecular perturbations. Here, using 3D-engineered muscle tissues in vitro and an mdx mouse model of DMD, we report that Rho-associated protein kinase (ROCK) contributes directly to this weakening process by reducing muscle quality and force but, unexpectedly, independent of differentiation or regeneration. We find that ARHGEF3, one of the RhoA guanine nucleotide exchange factors (GEFs), is elevated and responsible for RhoA/ROCK activation in mdx muscles and that depleting ARHGEF3 in mdx mice restores muscle quality and morphology without affecting regeneration. Conversely, overexpressing ARHGEF3 further compromises mdx muscle quality in GEF activity- and ROCK-dependent manner. Notably, ARHGEF3/ROCK inhibition exerted the effects by rescuing autophagy which is commonly impaired in dystrophic muscles. Taken together, our findings uncover a new pathological mechanism of DMD and the therapeutic potential of targeting ARHGEF3-ROCK signaling in DMD.