Type 2 diabetes is associated with defects in insulin signaling and the resulting abnormal glucose and lipid metabolism. The complexity of insulin signaling cascades is highlighted by the existence of multiple isoforms of target proteins implicated in metabolic and gene-regulatory events. We utilized siRNA to decipher the specific role of predominant insulin receptor substrates and Akt isoforms expressed in human skeletal muscle. Gene silencing revealed specialized roles of insulin signaling cascades to metabolic endpoints. IRS-1 and Akt2 were required for myoblast differentiation and glucose metabolism, whereas IRS-2 and Akt1 were dispensable. A key role of IRS-2 and Akt1 in lipid metabolism was revealed, highlighting reciprocal relationships between metabolic pathways. Unraveling the isoform-specific regulation of glucose and lipid metabolism by key elements along insulin signaling cascades through siRNA-mediated gene silencing in human tissues will facilitate the discovery of novel targets for the treatment of diabetes and related metabolic disorders.