The aim of this study was to explore the regulatory role of the PI3K/Akt pathway in adipogenic trans-differentiation of myoblasts. C2C12 myoblasts were cultured and, subsequently, induced for adipogenic trans-differentiation. During trans-differentiation, levels of phosphorylated PI3K (P85 and P55 subunits) were increased progressively with early differentiation, but significantly decreased during late-phase differentiation. There were no changes in Akt protein levels, but its phosphorylation levels changed similarly to those of PI3K. Wortmannin treatment of the cells efficiently suppressed PI3K/Akt activation, resulting in significant inhibition of adipogenesis and to varying extents, downregulated expression of several adipogenic genes (PPARγ, C/EBPα, FABP4 and FATP1). In addition, silencing the PI3K gene by siRNA transfection also inhibited adipogenic trans-differentiation in C2C12 cells. Moreover, suppressing both activation and expression of PI3K/Akt induced apoptosis. Taken together, our findings indicated that the PI3K/Akt pathway plays a key role in adipogenic trans-differentiation of myoblasts.