Cigarette smoking causes insulin resistance. However, nicotine induces anti-inflammation and improves glucose tolerance in insulin-resistant animal models. Here, we determined the effects of nicotine on glucose metabolism in insulin-sensitive C57BL/J6 mice. Acute nicotine administration (30 min) caused fasting hyperglycemia and lowered insulin sensitivity acutely, which depended on the activation of nicotinic-acetylcholine receptors (nAChRs) and correlated with increased catecholamine secretion, nitric oxide (NO) production, and glycogenolysis. Chlorisondamine, an inhibitor of nAChRs, reduced acute nicotine-induced hyperglycemia. qRT-PCR analysis revealed that the liver and muscle express predominantly β4 > α10 > α3 > α7 and β4 > α10 > β1 > α1 mRNA for nAChR subunits respectively, whereas the adrenal gland expresses β4 > α3 > α7 > α10 mRNA. Chronic nicotine treatment significantly suppressed expression of α3-nAChR (predominant peripheral α-subunit) in liver. Whereas acute nicotine treatment raised plasma norepinephrine (NE) and epinephrine (Epi) levels, chronic nicotine exposure raised only Epi. Acute nicotine treatment raised both basal and glucose-stimulated insulin secretion (GSIS). After chronic nicotine treatment, basal insulin level was elevated, but GSIS after acute saline or nicotine treatment was blunted. Chronic nicotine exposure caused an increased buildup of NO in plasma and liver, leading to decreased glycogen storage, along with a concomitant suppression of Pepck and G6Pase mRNA, thus preventing hyperglycemia. The insulin-sensitizing effect of chronic nicotine was independent of weight loss. Chronic nicotine treatment enhanced PI-3-kinase activities and increased Akt and glycogen synthase kinase (GSK)-3β phosphorylation in an nAChR-dependent manner coupled with decreased cAMP response element-binding protein (CREB) phosphorylation. The latter effects caused suppression of Pepck and G6Pase gene expression. Thus, nicotine causes both insulin resistance and insulin sensitivity depending on the duration of the treatment.