OBJECTIVE: In vivo evidence suggests that insulin entry into skeletal muscle is rate limiting for its overall metabolic action. Although there has been controversy regarding whether insulin crosses the endothelium by a passive (transcellular or paracellular) or mediated process, accumulating data favor the latter. Here, we addressed whether insulin signaling within the endothelial cell is required for the first step of transendothelial insulin transport: its uptake by the endothelial cell. RESEARCH DESIGN AND METHODS: Bovine aortic endothelial cells (bAECs) were incubated in serum-free medium for 6 h before addition of 50 nmol/l fluoroisothiocyanate (FITC)-labeled insulin for 30 min, and uptake of FITC insulin was quantified by confocal immunocytochemistry. RESULTS: Cellular insulin uptake was temperature dependent, being greater at 37 vs. 4 degrees C (P < 0.05). Inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin), mitogen-activated protein kinase kinase (MEK) (PD98059), the cSrc-family tyrosine kinase (PP1), or the insulin receptor tyrosine kinase (genistein) markedly diminished FITC insulin uptake (P < 0.05 for each). In contrast, inhibiting the phosphotyrosine phosphatase protein tyrosine phosphatase 1B further stimulated insulin uptake (P < 0.05). Addition of the inflammatory cytokine 5 ng/ml tumor necrosis factor-alpha (TNF-alpha) for 6 h before adding 50 nmol/l FITC insulin diminished insulin uptake significantly (P < 0.05). This inhibitory effect of TNF-alpha could be partially reversed by a specific p38 MAPK inhibitor (SB203580). CONCLUSIONS: Insulin uptake by bAECs requires intact insulin signaling via both the PI 3-kinase and MEK signaling cascades and the cSrc-family tyrosine kinases, and endothelial cell insulin uptake is sensitive to cytokine-induced insulin resistance.