Objective: To determine the anticancer potential of the methanolic extract from Ephedra alata against breast cancer both in vitro and in vivo. Methods: The effects of the methanolic extract of Ephedra alata on the viability, migration as well as apoptosis of breast cancer 4T1 cells were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay, Transwell assay, and annexin V-FITC staining assay, respectively. Histological examination was also carried out. Moreover, a murine breast cancer model was established to evaluate the inhibitory effect of the extract. Biochemical parameters including hepatic and non-hepatic enzymes, malondialdehyde, and glutathione were investigated. Results: The methanolic extract of Ephedra alata showed a strong anti-proliferative and anti-migratory activity against 4T1 cells in a dose-dependent manner. It also induced apoptosis in 4T1 cells. In an in vivo mouse model, the extract markedly inhibited tumor growth, reduced malondialdehyde, and hepatic and non-hepatic enzymes as well as increased glutathione level. Conclusions: The methanolic extract of Ephedra alata inhibits breast cancer in vitro and in vivo, which may be a promising anticancer agent.