Many cytokines increase their receptor affinity for Janus kinases (JAKs). Activated JAK binds to signal transducers and activators of transcription, insulin receptor substrates (IRSs), and Shc. Intriguingly, insulin acting through its own receptor kinase also activates JAK2. However, the impact of such activation on insulin action remains unknown. To determine the contribution of JAK2 to insulin signaling, we transfected L6 myotubes with siRNA against JAK2 (siJAK2), reducing JAK2 protein expression by 75%. Insulin-dependent phosphorylation of IRS1/2 and Shc was not affected by siJAK2, but insulin-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) extracellular signal-related kinase, p38, and Jun NH 2 -terminal kinase and their respective upstream kinases MKK1/2, MKK3/6, and MKK4/7 was significantly lowered when JAK2 was depleted, correlating with a significant drop in insulin-mediated cell proliferation. These effects were reproduced by the JAK2 inhibitor AG490. Conversely, insulin-stimulated Akt phosphorylation, glucose uptake, and GLUT4 translocation were not affected by siJAK2. Interestingly, in two insulin-resistant states, siJAK2 led to partial restoration of Akt phosphorylation and glucose uptake stimulation but not of the MAPK pathway. These results suggest that JAK2 may depress the Akt to glucose uptake signaling axis selectively in insulin-resistant states. Inhibition of JAK2 may be a useful strategy to relieve insulin resistance of metabolic outcomes. Diabetes 55: 942-951, 2006 P olypeptides such as erythropoietin, prolactin, leptin, angiotensin, growth hormone, most interleukins, and interferon-␥ bind to receptors that lack intrinsic kinase activity, recruiting and activating cytoplasmic tyrosine kinases of the Janus family (JAK) consisting of JAK1, JAK2, JAK3, and Tyk2 (1-3). Activated JAK phosphorylates tyrosine residues within itself and the associated receptor forming high-affinity binding sites for a variety of signaling proteins containing Src homology 2 and other phosphotyrosine-binding domains, including signal transducers and activators of transcription, insulin receptor substrates (IRSs), and the adaptor protein Shc (1-4). Shc is responsible for the activation of the mixed-function tyrosine/serine/threonine kinases MEKs (mitogen-activated protein kinases [MAPKs] that in turn activate the extracellular signalrelated kinanes [ERKs]) MAPKs, important for the mitogenic response (4). JAK is also the mediator of insulin-like effects of some polypeptides like growth hormone through phosphorylation of IRS and activation of their downstream effectors phosphatidylinositol (PI) 3-kinase and Akt, mainly required for metabolic functions (5-8). Moreover, JAK is the cytoplasmic kinase through which circulating factors such as tumor necrosis factor-␣ and pro-inflammatory cytokines negatively regulate insulin action through serine phosphorylation of IRS-1 or induction of suppressors of cytokine signaling (SOCS) expression (9).Insulin increases the autophosphorylation and tyrosine kinas...