Vascular smooth muscles (VSMCs) are known to be the key drivers of intimal thickening which contribute to early progression of atherosclerosis. VSMCs are the major producers of extracellular matrix within the vessel wall and in response to atherogenic stimuli they could modify the type of matrix proteins produced. Serotonin receptor 2B (5-HT2B receptor/HTR2B) has been implicated in several chronic fibrotic and vascular diseases. Although studies have successfully demonstrated the efficacy of HTR2B blockade in attenuating fibrotic disease, the role of 5-HT2B receptor in TGFβ mediated VSMC differentiation remain largely unknown. In the present study, we investigated the potential of targeting the 5-HT2B receptor to prevent TGFβ induced VSMCs differentiation. Our results showed that 5-HT2B receptors are expressed in human atherosclerotic lesion and HTR2B expression positively correlated to the VSMCs markers. We show that AM1125, a selective 5-HT2B receptor inhibitor, significantly inhibits TGFβ1 induced production of collagen and CTGF. The investigation of underlying mechanisms indicated that 5-HT2B receptor antagonism blocks phospho-Smad2 mediated downstream signaling of TGFβ1 in vascular smooth muscle cells. Collectively, the HTR2B/TGF-β1/Phospho-Smad2 pathway plays a critical role in the regulation of VSMCs differentiation. Our findings might serve 5-HT2B receptor as a therapeutic target to limit TGF-β1 induced VSMC differentiation.