Colistin is often used as a last resort for treating multidrug-resistant infections, particularly in critically ill patients in intensive care units. Nonetheless, its side effects, including myopathy, require careful monitoring. Vasoconstrictive drugs are also used in intensive care to increase blood pressure and improve blood flow to vital organs, which can be compromised in critically ill patients. The exact mechanism of colistin-induced muscle toxicity is of significant interest due to its potential intensive-care clinical implications. Colistin alone or in combination with vasoconstrictive agents was administrated in non-septic and LPS-induced septic animals for 10 days. Histopathological evaluation of the gastrocnemius muscle and dot-blot protein tissue analysis were performed. Increased intramuscular area, de-organization of the muscle fibers and signs of myopathy were observed in colistin-treated animals. This effect was ameliorated in the presence of vasoconstrictive drugs. Administration of colistin to septic animals resulted in a decrease of AMPK and cyclin-D1 levels, while it had no effect on caspase 3 levels. Vasoconstrictive drugs’ administration reversed the effects of colistin on AMPK and cyclin D1 levels. Colistin’s effects on muscle depend on septic state and vasoconstriction presence, highlighting the need to consider these factors when administering it in critically ill patients.