Phosphoinositide 3-kinase (PI3K) is a principal regulator of Akt activation and myogenesis; however, the function of PI3K p110β in these processes is not well defined. To address this, we investigated the role of p110β in Akt activation and skeletal muscle cell differentiation. We found that Akt phosphorylation was enhanced in p110β-deficient myoblasts in response to Insulin-like Growth Factor-I (IGF-I), epidermal growth factor, or p110α overexpression, as compared to p110β-sufficient cells. This effect was associated with increased mammalian target of rapamycin complex 2 activation, even in myoblasts deficient in mSin1 and rictor. Conversely, in response to the G-protein-coupled receptor agonist lysophosphatidic acid, Akt phosphorylation was attenuated in p110β-deficient myoblasts. Loss of p110β also enhanced the expression of myogenic markers at the myoblast stage and during the first 48 h of differentiation. These data demonstrate that reductions in p110β are associated with agonist-specific Akt hyperactivation and accelerated myogenesis, thus revealing a negative role for p110β in Akt activation and during myoblast differentiation.