Muscle atrophy, resulting from physical inactivity or protein deficiency, is a significant health concern. beta-hydroxy-beta-methylbutyrate (HMB) has potential in preserving muscle mass, but its mechanisms in various atrophy-inducing conditions are not fully understood. This study aimed to investigate HMB's effects on muscle atrophy induced by inactivity and protein deprivation, and to elucidate the underlying molecular mechanisms. Rats were subjected to inactivity or protein-deficient diets with or without HMB supplementation. Muscle morphology, strength, and biochemical parameters were assessed. In vitro studies using C2C12 myoblasts and mouse skeletal muscle satellite cells exposed to interleukin-6 (IL-6) explored molecular pathways involved in HMB's protective effects. Inactivity and protein deprivation led to muscle atrophy, reduced strength, and altered biochemical markers. HMB supplementation partially mitigated these effects, preserving muscle mass and function. HMB attenuated atrophy markers (Muscle Atrophy F-box and Muscle RING Finger 1 (MuRF1)) and maintained myogenic factor (Myogenin (MyoG)) levels. In vitro studies revealed that HMB's protective effects were mediated through the AKT/mTOR pathway, with concurrent regulation of autophagy pathways and preservation of mitochondrial function in both myoblasts and satellite cells. HMB specifically protected satellite cell viability and function through AKT-dependent mechanisms, maintaining protein synthesis and reducing apoptosis under IL-6-induced stress conditions. HMB supplementation shows protective effects against muscle atrophy induced by inactivity and protein deprivation, through multiple mechanisms including AKT/mTOR pathway activation, autophagy regulation, and maintenance of mitochondrial function in both myoblasts and satellite cells. These findings suggest HMB as a potential therapeutic strategy for preventing muscle atrophy in various clinical scenarios.