Objective To confirm whether fasudil can block C2C12 myoblasts respiration dysfunction triggered by Rho-associated coiled-coil containing protein kinase 1 (ROCK1), and whether it can block the occurrence of muscle atrophy. Methods C2C12 myoblasts were cultured in vitro, and 2% horse serum was used to induce cell differentiation and maturation. The obtained mature muscle tubule cells were divided into four groups according to the different stimuli: Ad-GFP group, only transfected GFP-adenovirus vector (Adv) in C2C12 myoblasts; Ad-ROCK1 group, transfected ROCK1-Adv in C2C12 myoblasts to induce ROCK1 overexpression; Ad-GFPF group, transfected GFP-Adv and given 10 μmol/L fasudil in C2C12 myoblasts; and Ad-ROCK1F group, transfected ROCK1-Adv and given 10 μmol/L fasudil in C2C12 myoblasts. The oxygen consumption rate (OCR) and extracelluar acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by cell energy metabolism analyzer (Seahorse), so as to determine the effect of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts. Mitochondrial fission was measured by MitoTracker^® red fluorescent probes. The expressions of ROCK1, mitochondrial-related protein 1 (Drp1) and phosphorylated p-Drp1, E3 ubiquitin ligase muscle RING finger-1 protein (MuRF1) and muscle atrophy F-box (MAFbx, Atrogin 1) was measured by Western blotting analysis. Results Seahorse analysis showed that the OCR, ECAR, basal respiration, maximal respiration and respiration required for coupling ATP of C2C12 myoblasts in the Ad-ROCK1 group were significantly increased compared with those in the Ad-GFP group (P<0.01); Meanwhile, MitoTracker^® staining showed that the mitochondrial fission was increased and the mitochondrial size frequency distribution shifted left in the Ad-ROCK1 group. After exposed to fasudil, the OCR and EACR of C2C12 myoblasts in the Ad-ROCK1F group were significantly decreased versus the Ad-ROCK1 group, and the basal respiration and maximal respiration were significantly increased (P<0.05). Western blotting analysis showed that p-Drp1/Drp1 ratio, and the expressions of ROCK1, MuRF1 and Atrogin1 in Ad-ROCK1F group were significantly reduced compared with Ad-ROCK1 group (P<0.05). Conclusion Fasudil, an inhibitor of ROCK1, can block the abnormal cell respiration of C2C12 myoblasts caused by overexpressed ROCK1 in vitro, and can reduce the activity of mitochondrial kinetic protein and the expression of muscle atrophy-related proteins.