To improve the bioavailability of buckwheat protein， buckwheat antioxidant peptides (BAPs) was isolated from the fermentation hydrolysate of buckwheat by using Sephadex G-25 and reversed phase-high performance liquid chromatography (RP-HPLC)， and its structure and composition were preliminarily analyzed. The H2O₂-induced RAW 264.7 macrophages cell model of oxidative stress was further established to evaluate the antioxidant activity of BAPs. The results showed that the BAPs obtained after separation by RP-HPLC has a purity of 96.25% and a molecular weight of 5.8 ku. Vitamin C equivalent antioxidant capacity (VCEAC) was used to indicate that DPPH·， ABTS+·， ·OH scavenging ability and reducing power of BAPs were (12.12±0.27)， (153.82±5.04)， (901.95±39.30)， (109.23±1.18) μg/mg， respectively. In the RAW 264.7 macrophage oxidative stress model， BAPs with the concentration of 200 μg/mL had a significant (P<0.05) effect on all measured antioxidant indicators compared with the model group， which significantly (P＜0.05) promoted the viability of oxidatively damaged cells by 13.80%， reduced the levels of intracellular reactive oxygen species (ROS) by 40.63% and malondialdehyde (MDA) by 11.92 nmol/mg prog. Meanwhile， the activities of superoxide dismutase(SOD) and catalase (CAT) were increased by 23.48 and 15.71 U/mg prot at this concentration， respectively. The above results showed that BAPs can effectively inhibit the oxidative stress response of RAW 264.7 macrophages， which could provide a theoretical basis for the development of buckwheat peptides as natural antioxidants.