Chronic subdural hematoma is identified as an intracranial hemorrhagic disorder of human. Presently, neither surgical nor symptomatic treatment of Western medicine has achieved good curative effects. This study aimed to clarify efficacy of Peiyuan-Huayu formula on neovascularization and explore associated mechanisms. Rats were intragastricly administrated with Peiyuan-Huayu formula and drug containing serum was harvested. Cell counting kit 8 assay was used to determine proliferation of human microvascular endothelial-1 cells. Human microvascular endothelial-1 cells were divided into negative group, blank-serum group and drug-serum group. In vitro scratch healing assay was employed to evaluate migration distance of human microvascular endothelial cells-1. Tube-like structures formation based on human microvascular endothelial-1 cells was conducted using tube formation assay on matrigel. Expression of vascular endothelial growth factor A, mitogen-activated protein kinase kinase, phosphorylation of mitogen-activated protein kinase kinase and phosphorylation of extracellular signal-regulated kinase was determined with western blotting. The 15 % drug-serum is optimal concentration for inhibiting human microvascular endothelial cells-1 proliferation. Peiyuan-Huayu formula drug-serum remarkably inhibited migration of human microvascular endothelial cells compared to Blank-serum group (p<0.05). Peiyuan-Huayu formula drug-serum obviously suppressed angiogenesis compared to blank-serum group (p<0.05). Peiyuan-Huayu formula drug-serum down-regulated vascular endothelial growth factor A expression, activated phosphorylation of mitogenactivated protein kinase kinase and induced phosphorylation of extracellular signal-regulated kinase 1/2 in human microvascular endothelial-1 cells. In conclusion, Peiyuan-Huayu formula inhibited proliferation, migration and angiogenesis of human microvascular endothelial-1 cells. Effects of Peiyuan-Huayu formula were mediated by reducing expression of mitogen-activated protein kinase kinase 1/2, phosphorylation of mitogen-activated protein kinase kinase 1/2, phosphorylation of extracellular signal-regulated kinase 1/2 and vascular endothelial growth factor molecule.