The effect of beta-hydroxy-beta-methylbutyrate (HMB) supplementation associated with exercise training at different intensities and frequencies on skeletal muscle regeneration of muscle-injured rats was investigated. Male Wistar rats were divided into sedentary and trained groups. The sedentary groups were subdivided into non-injured (SED-Ct), non-injured supplemented with HMB (SED-Ct-HMB), injured (SED), and injured with HMB (SED-HMB), and the trained groups were injured, supplemented with HMB, and then divided into training three times a week without load (HT3) or with load (HT3L) and training five times a week without load (HT5) and with load (HT5L). The rats received a daily dose of HMB associated with 60 min of swimming with or without 5% body mass load for 14 days. On the 15th day, cryoinjury was performed in the right tibialis anterior muscle (TA), and 48 h later, supplementation and training continued for 15 days. After the last session, the TA was dissected and a cross-sectional area (CSA) of muscle fibers was used to determine the percentage of CSA fibers and connective tissue (%CT), as well as the total and phosphorylated protein contents. SED-HMB showed increased CSA and decreased %CT and TGF-β when compared to SED. HT3 showed increased CSA and reduced %CT accompanied by increased IGF-1/Akt, myogenin, and MuRF1, and decreased TGF-β. The CSA of HT5L also increased, but at the cost of a higher %CT compared to the other groups. Our results demonstrated that HMB associated with training without load and with lower frequency per week may be a valuable strategy for skeletal muscle regeneration.