Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines if myonuclear accretion and myogenic differentiation are required, and that Insulin-like Growth Factor-I (IGF-I)/Akt/Glycogen Synthase Kinase 3 (GSK-3) signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (-40%) and plantaris muscle (-27%). Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression which coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3 were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain perinatal, markers of myoblast proliferation and differentiation, and a strong induction of Muscle Regulatory Factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I induced MRF expression is facilitated by inactivation of GSK-3 and selectively occurs in the myoblast population.This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3.