Aim To study the changes of gut microbiota of hyperlipemia guinea pigs by microbial 16S rDNA sequencing. Methods Guinea pigs were randomly divided into normal group and model group, with 10 in each group. Guinea pig models of hyperlipidemia were established by high fat diet. After feeding for 8 weeks, serum total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDLC) and high density lipoprotein cholesterol (HDLC) levels were detected. HE staining was performed to detect the histological changes of liver, aorta and colon. The changes of gut microbiota were detected by 16S rDNA analysis. Results The levels of TC, TG, LDLC and HDLC of model group were higher than that of control group (P<0.01). According to the histomorphological test, all guinea pigs in the model group showed severe fatty liver, but only one of them appeared early symptoms of atherosclerosis. The gut microbiota 16S rDNA high-throughput sequencing analysis result showed that, compared with control group, at the phyla level, the proportion of Synergistetes was obviously increased (P<0.05); at the genus level, the proportions of lachnospiraceae NK4A136, ruminococcus, helicobacter, odoribacter, allobaculum and caldicoprobacter of the model group were significantly reduced (P<0.05); the proportions of fretibacterium, lachnospiraceae XPB1014, pyramidobacter and enterorhabdus were obviously increased (P<0.05). Conclusions The compositions and proportions of gut microbiota in guinea pigs were significantly changed by high-fat feeding. The results of the experiment laid a theoretical foundation for the study of the mechanism of hyperlipidemia based on intestinal flora.