Accumulating evidence has suggested that medium-, long-, and medium-chain (MLM) structured lipids have anti-obesity effects, but whether they can alleviate the development of atherosclerosis (AS) and affect the composition of the gut microbiota in high-fat diet-fed ApoE-/- mice has not been elucidated. The present study found that MLM structured lipid supplementation could significantly decrease obesity-related parameters compared with high-fat diet alone in ApoE-/- mice. Additionally, MLM structured lipids could significantly decrease the blood glucose and increase the serum total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) levels. Additionally, high-dose MLM structured lipids supplementation could reduce the area of atherosclerotic lesions and decrease the expression of VCAM-1, MCP-1 and CD68, which are related to inflammation in aortic tissue. Further analysis showed that MLM structured lipids could significantly reduce lipid accumulation in the adipose tissue of high-fat diet-fed ApoE-/- mice. The relative protein expression of SREBP-1, ACC, FAS, C/EBPα and PPARγ was decreased and the ratio of p-AMPK/AMPK was increased in epididymis white adipose tissue (eWAT) after MLM structured lipids treatment. Additionally, MLM structured lipids supplementation regulated the bacterial composition, including reducing the Firmicutes/Bacteroidetes ratio, increasing the relative abundance of short-chain fatty acid-producing bacteria (Blautia and Anaerotruncus), decreasing the relative abundance of [Ruminococcus] torques group, Ruminiclostridium 9, Catenibacterium and [Eubacterium] fissicatena group. Spearman's correlation analysis revealed significant correlations between changes in the gut microbiota and atherosclerosis-related indices. The results demonstrated that the alleviating effects of MLM structured lipids supplementation on AS in high-fat diet-fed ApoE-/- mice were closely related to reshaping the composition of the gut microbiota.