Sex hormone binding globulin (SHBG) is a hepatic glycoprotein often reduced under insulin resistance and hepatic steatosis. Nowadays, the role of glucose and insulin (HI) on hepatic SHBG expression and secretion is still unclear. Therefore, the aim of this study has been to investigate the effects of growing doses of glucose in the absence or presence of 100 nM HI for 48 and 96 h on SHBG synthesis in HepG2 cell line. After treatment, cells were lysed to evaluate protein expression and activation of insulin receptor (IRβ) and Akt, as well as the protein expression of SHBG and its master regulators (i.e., HNF4α and SIRT2) by immunoblotting. The secretion of SHBG in culture medium was measured with ELISA. High glucose, but not equimolar quantities of mannitol, led to increased IRβ protein expression in HepG2 as compared to normal condition. Conversely, cells co-treated with HI and high glucose, as well as with equimolar doses of mannitol, displayed reduced IRβ protein levels as compared to cells treated without HI. In these setting, an impaired insulin sensitivity in terms of p-IRβTyr1150/1151 and p-AktS473 was observed after acute insulin stimulation. A dose-dependent increase in SHBG protein expression, but not in its secretion, was observed after incubation with glucose alone; however, in the presence of HI a significant reduction in SHBG protein expression and secretion at high glucose was observed as compared to condition without HI. A reduction in both HNF4α and SIRT2 protein expression was observed after co-stimulation with high glucose and HI. Moreover, high levels of glucose and HI also limited the triiodothyronine-induced upregulation of SHBG expression and secretion. In conclusion, these results demonstrated that hyperglycemia and hyperinsulinemia induced insulin resistance in association with a down-regulation of SIRT2, HNF4α and SHBG expression and secretion. Under these conditions hepatic cells may became less responsive to physiological stimuli to the production of SHBG. Supported by Fondazione Cianciola.