Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA2) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA2 are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA2 and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA2 by 1.5-fold ( p < 0.01), which was blocked by the cPLA2 inhibitor MAFP (10 microM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 microM; 15 min) and ERK1/2 inhibitor PD98059 (20 microM; 15 min) abolished the INS-mediated increase in cPLA2 phosphorylation by 59% (p < 0.001), and by 75% ( p < 0.001), respectively. Further, inhibition of cPLA2 with cPLA2 inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% ( p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% ( p < 0.001). The effect of INS on VSMCs proliferation was significantly ( p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA2 and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA2 activity.