Insulin resistance and smoking are significant risk factors for cardiac and cerebral vascular diseases. Because vascular smooth muscle cells (VSMCs) play a key role in the development and progression of atherosclerosis, we investigated the effect of nicotine on insulin-induced mitogenic signaling in aortic VSMCs isolated from Sprague Dawley rats. RT-PCR revealed the expression of 2-7, 10, 1-3, , and subunits of the nicotinic acetylcholine receptor (nAChR) in the cells. Short-term nicotine treatment stimulated phosphorylation of p44/42-MAP kinase, p38-MAP kinase, and STAT3. However, an additive effect of nicotine pretreatment on insulin stimulation was only observed on p44/42-MAP kinase. The nicotine-induced phosphorylation of p44/42-MAP kinase and [methyl-³H] thymidine incorporation were effectively suppressed by a 7-nAChR-selective antagonist, methyllycaconitine, and the phosphorylation of p44/42-MAP kinase was stimulated by a 7-nAChR-specific agonist, GTS21. Furthermore, the phosphorylation was mediated via calmodulin kinase II, Src, and Shc. Interestingly, long-term (48 h) pretreatment with nicotine increased the amount of 7-AChR in the high-speed pellet containing plasma membrane fraction, and insulin-induced phosphorylation of p44/42-MAP kinase. These results provide the first evidence that acute exposure to nicotine enhances insulin-induced mitogenesis predominantly by affecting the phosphorylation of p44/42-MAP kinase, and that chronic exposure further augments the insulin signal via up-regulation of 7-nAChR which may be crucial for the development and progression of atherosclerosis in large vessels.