Vascular smooth muscle cells (VSMCs) are an important component of arterial walls, and their dysfunction may serve an important role in the development of cardiovascular diseases, including atherosclerosis and restenosis. Paeoniflorin (PF) is a principal component of the commonly used traditional Chinese medicine, peonies. To the best of our knowledge, the effects of PF on apoptosis and proliferation of VSMCs and its underlying molecular mechanisms have not been widely reported. Therefore, the present study was designed to investigated this phenomenon. VSMCs were treated with different concentrations of PF (25, 50 and 100 µg/ml) for 12, 24 or 48 h. The data demonstrated that PF treatment not only significantly decreased cell viability and DNA synthesis but also blocked G0/G1 cell cycle progression. This effect was associated with a decreased expression of cyclin D1, cyclin E, cyclin‑dependent kinase (CDK)4 and CDK2 as well as an upregulation of p21. Notably, a significant concentration‑dependent decrease in the phosphorylation of p65 and nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitor‑α (IκBα) was observed. In addition, it was demonstrated that PF promoted the apoptosis of VSMCs, which was associated with the increased expression of caspases. In conclusion, PF inhibited the proliferation of VSMCs by downregulating proteins associated with the nuclear factor‑κB signaling pathway. Furthermore, it promoted the apoptosis of VSMCs by upregulating the expression of caspases. These results may be useful in improving the understanding of the molecular mechanisms underlying the apoptotic and anti‑proliferative effects of PF on VSMCs, and facilitate the development of novel treatments for cardiovascular diseases.