In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK)pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD98059 and U0126, which reduced insulin-stimulated glucose uptake by approximately 33% and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKC/, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4-myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.