Insulin regulates glucose metabolism and activates cell growth. As the respiratory system is an alternative route for insulin administration in patients with diabetes mellitus, we studied the effect of insulin on rabbit tracheal airway smooth muscle (ASM) cell proliferation. We show that treatment of quiescent ASM cells with insulin for 24 h increased cell number compared to control cells. This increase was similar to the increase caused by the addition of fetal bovine serum (FBS). Moreover, in contrast to cells treated with FBS, exposure of ASM cells with insulin for longer periods (48 and 72 h) did not have any further influence on cell proliferation. In ASM cells, insulin activated the phosphatidylinositol 3-kinase (PI3 K) pathway, while FBS activated both PI3 K and the mitogen-activated protein kinase (MAPK) pathway. The PI3K pathway inhibitors LY294002 and wortmannin abolished the stimulation of cell proliferation by insulin, indicating a role for this pathway in the cellular response to insulin. These results show that while insulin has a mitogenic effect on ASM cells, prolonged insulin treatment does not lead to excessive ASM cell proliferation and suggest that the use of aerosolized insulin does not represent a potential hazard for airway remodeling.