Konjac mannan oligosaccharides (KMOS) have a variety of beneficial effects on human health, but their regulator effects on atherosclerosis (AS) and the involved molecular mechanisms have not been well understood. In this study, AS model was established by feeding ApoE-/- mice with high fat diet (HFD) for 14 weeks and used for investigating the effects of KMOS intervention on aortic lipid accumulation, serum lipid, and inflammatory factors. The regulatory effect of KMOS on gut microbiota of AS mice was analyzed by using Illumina high-throughput sequencing technology. The results showed that KMOS supplementation significantly inhibited the increase of body and liver weight induced by HFD. Comparing with HFD group, the area ratio and total area of plaque lesions in aortic sinus induced by HFD was reduced by 57.4% and 57.9%, respectively after KMOS treatment. The total serum cholesterol levels and low-density lipoprotein cholesterol levels were decreased by 21.1% and 31.1%, whereas the high-density lipoprotein cholesterol level was significantly increased by 63.0% after KMOS treatment. Meanwhile, lipid accumulation in the liver of HFD induced ApoE-/- mice was significantly improved. The levels serum inflammatory factors (tumor necrosis factor α, interleukin-6, interleukin-1β, and monocyte chemotactic protein-1) were reduced by KMOS, compared with AS model group. Moreover, the expression of genes associated with liver cholesterol reverse transport was significantly increased by KMOS. Furthermore, KMOS inhibited the dysbiosis of gut microbiota in ApoE-/- mice induced by HFD with increased relative abundances of g_norank_f_Lachnospiraceae, g_Alistipes, and g_norank_f_Ruminococcaceae. The production of short chain fatty acids in the cecum was promoted and the expression of intestinal tight junction proteins was increased by KMOS to prevent the impairment of intestinal barrier function. Thus, KMOS could alleviate the development of AS in ApoE-/- mice induced by HFD through the regulation of cholesterol metabolism and gut microbiota. This results aimed to provide theoretical basis for development of KMOS related functional food.