The insulin-like growth factors (IGF-I and -II), working through the type-1 IGF receptor (IGF-1R), are key mediators of skeletal muscle fiber growth and hypertrophy. These processes are largely dependent upon stimulation of proliferation and differentiation of muscle precursor cells, termed myoblasts. It has not been rigorously determined whether the IGFs can also mediate skeletal muscle hypertrophy in a myoblast-independent fashion. Similarly, although both the phosphatidylinositol 3-kinase (PI3-K) and calcineurin signaling pathways have been implicated in skeletal muscle hypertrophy, these pathways are also involved in skeletal myoblast differentiation. To determine if the IGFs can stimulate skeletal muscle hypertrophy in a myoblast-independent fashion, a retroviral expression vector which mediated overexpression of the human IGF-1R in rat L6 skeletal myotubes (immature muscle fibers), but not in myoblasts, was developed and validated. Compared to controls, L6 myotubes transduced with this vector accumulated significantly higher amounts of myofibrillar proteins in a ligand- and receptor-dependent manner, accompanied by significantly increased rates of protein synthesis. The stimulation of myotube hypertrophy was independent of myoblast contributions, as these cultures did not exhibit increased levels of myoblast proliferation or differentiation. Experiments with PI3-K and calcineurin inhibitors indicated myoblast-independent myotube hypertrophy was mediated by PI3-K, but not calcineurin, signaling. This study demonstrates IGF can mediate skeletal muscle hypertrophy in a myoblast-independent fashion, and suggests that muscle-specific overexpression of the IGF-1R or stimulation of its signaling pathways could be used to develop strategies to ameliorate muscle wasting without stimulating proliferative pathways leading to carcinogenesis or other pathologic sequelae.