Paeonol (2'-hydroxy-4'-methoxyacetophenone) is the major active compound of Mautan cortex and has been demonstrated to inhibit platelet aggregation in previous studies. The current study aimed to elucidate the underlying molecular mechanism of paeonol in recanalizing thrombi. The presence of indicators of prothrombotic state (PTS) in the serum of the model animals were determined by enzyme‑linked immunosorbent assay (ELISA) assay and the cytotoxicity of paeonol on human umbilical vein endothelial cell (HUVEC) cultures was estimated by 3‑(4,5 dimethylthiazol‑2‑yl)-2,5-diphenyltetrazolium bromide assay. The possible underlying signaling pathway involved in the interaction between paeonol and vascular endothelial growth factor 165 (VEGF165) was investigated using western blotting. The levels of 6‑keto‑prostaglandin F1α, fibronectin, and VEGF165 in serum were significantly upregulated by the treatment of paeonol while the levels of fibrinogen, D‑dimer, and thromboxane B2 were significantly downregulated (P<0.05). With increased paeonol concentration, the cell viability of HUVECs gradually decreased. The results of the western blot analysis demonstrated that paeonol increased the expression levels of phosphorylated‑extracellular signal‑regulated kinase (ERK1/2) and VEGF165 but had no marked effect on the expression level of ERK1/2. Paeonol has the potential to improve PTS and recanalize thrombi in animal models, which may be by the upregulation of VEGF165 via the ERK1/2 mitogen activated protein kinase signaling pathway. However, this positive effect depended on the concentration of paeonol used, an unsuitably high concentration of the compound exerted negative effects on the anti‑thrombosis signaling pathways.