Macauba (Acrocomia aculeata) is a palm tree native from Brazil, who pulp is rich in oil that has a high content of oleic acid and carotenoids. Macauba pulp oil can bring health benefits due to its bioactive compounds, however, its effects on gut health are unknown. Thus, the objective of this study was to evaluate the effect of macauba pulp oil in the intestinal health in mice fed a high-fat diet. Male C57BL1/6 mice were randomly divided into 3 groups (10 animals/group): control diet (CD), high-fat diet (HF) and high-fat diet with 4% of macauba pulp oil (HFM). Short-chain fatty acid, fecal pH, and histomorphometric analysis of the colon were performed. Content of colon samples were used on microbiome analysis using 16S rRNA amplicon sequencing. Animals from the HFM group had higher butyric acid content and goblet cells number, greater circular and longitudinal muscle layer, and higher alpha-diversity compared with the HF group. Moreover, consumption of MPO reduced Desulfobacterota phylum, Ruminococcaceae, Oscillospiraceae, Prevotellaceae, Bifidobacteriaceae family, Faecalibacterium, Prevotella, Ruminococcus and Enterorhabdus genus. Therefore, macauba pulp oil was able to modulate the gut microbiota and enhance intestinal barrier morphology, showing preventive effects on gut dysbiosis in mice fed a high-fat diet.