Vascular smooth muscle cells (VSMCs) constitute the major cellular component of the vessel tunica media. VSMC proliferation is a key feature in developing vessels and pathological states such as atherosclerosis and restenosis. Transforming growth factor (TGF)-beta is a key regulator of VSMCs, but its effect on VSMC proliferation and apoptosis are controversial. Here, we characterized TGF-beta effects on basal-, serum-, and platelet-derived growth factor-BB-induced primary mouse VSMC proliferation. TGF-beta led to potent growth inhibition of VSMCs isolated from normal mouse aortae without inducing apoptosis. Growth inhibition by TGF-beta was due to G0/G1 arrest. Next, we explored distinct signaling pathways activated by TGF-beta and the effects of pharmacological inhibition of these. TGF-beta led to activation of Smad2/3, p38, p42/44, and c-Jun NH2-terminal kinase (JNK) pathways, assessed by phosphorylation, immunofluorescence, and reporter gene analysis. TGF-beta-dependent growth inhibition was specifically attenuated by pharmacological blockade of the TGF-beta type I receptor (TbetaRI) kinase or p38 mitogen-activated protein kinase pathways, whereas blockade of p42/44 or JNK kinases did not influence the effect of TGF-beta. TbetaRI kinase inhibition blocked all downstream pathways including Smad and p38 phosphorylation. In contrast, p38 inhibition did not alter Smad function, as assessed by translocation or reporter gene expression, but selectively inhibited p38 activity. These results demonstrate that TGF-beta acts as a potent antiproliferative mediator in VSMCs, irrespective of the proliferative stimulus, without inducing apoptotic effects. The anti-proliferative effect of TGF-beta is due to G0/G1 arrest and mediated primarily by the p38 pathway, suggesting that p38 kinase is central to TGF-beta-mediated growth inhibition in primary mouse VSMCs.