In the past decade, the high morbidity and mortality of atherosclerotic disease have been prevalent worldwide. High-fat food consumption has been suggested to be an overarching factor for atherosclerosis incidence. This study aims to investigate the effects of kefir peptides on high-fat diet (HFD)-induced atherosclerosis in apolipoprotein E knockout (ApoE-/-) mice. 7-week old male ApoE-/- and normal C57BL/6 mice were randomly divided into five groups (n = 8). Atherosclerotic lesion development in ApoE-/- mice was established after fed the HFD for 12 weeks compared to standard chow diet (SCD)-fed C57BL/6 and ApoE-/- control groups. Kefir peptides oral administration significantly improved atherosclerotic lesion development by protecting against endothelial dysfunction, decreasing oxidative stress, reducing aortic lipid deposition, attenuating macrophage accumulation, and suppressing the inflammatory immune response compared with the HFD/ApoE-/- mock group. Moreover, the high dose of kefir peptides substantially inhibited aortic fibrosis and restored the fibrosis in the aorta root close to that observed in the C57BL/6 normal control group. Our findings show, for the first time, anti-atherosclerotic progression via kefir peptides consumption in HFD-fed ApoE-/- mice. The profitable effects of kefir peptides provide new perspectives for its use as an anti-atherosclerotic agent in the preventive medicine.