Hyper Insulinemia is known to promote the progression/worsening of insulin resistance. Evidence reveals a hidden cost of hyperinsulinemia on plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2)-regulated filamentous actin (F-actin) structure, components critical to the normal operation of the insulin-regulated glucose transport System. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes PIP2/F-actin dysregulation and subsequent insulin resistance. Increased glycosylation events were detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nM insulin; 12 h) and in cells in which HBP activity was amplified by 2 mM glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP2 and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP2 corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP2/F-actin structure, pharmacological inhibition of the rate-limiting HBP enzyme [glutamine-fructose-6-phosphate amidotransferase (GFAT)] restored PIP2-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of GFAT was associated with a loss of detectable PM PIP2 and insulin sensitivity. Even less invasive challenges with glucose, in the absence of insulin, also led to PIP2/F-actin dysregulation. Mechanistically we found that increased HBP activity increased PM cholesterol, the removal of which normalized PIP 2/F-actin levels. Accordingly, these data suggest that glucose transporter-4 functionality, dependent on PIP2 and/or F-actin status, can be critically compromised by inappropriate HBP activity. Furthermore, these data are consistent with the PM cholesterol accrual/toxicity as a mechanistic basis of the HBP-induced defects in PIP2/F-actin structure and impaired glucose transporter-4 regulation. Copyright © 2011 by The Endocrine Society.