Macauba (Acrocomia aculeata) has aroused interest in the food industry due to the high nutritional value of its fruits. This study aimed to evaluate the protein quality and influence on biochemical markers, short chain fatty acids content, intestinal morphology, and intestinal functionality in Wistar rats of macauba kernel. Male young rats were divided into three groups (n = 8) that received a control diet (casein), and two test diets (M30: 30% semi-defatted macauba kernel flour or M50: 50% semi-defatted macauba kernel flour) for 29 days. Protein Efficiency Ratio (PER), Net Protein Ratio (NPR), True Digestibility (TD), biochemical, and intestinal morphology and functionality markers were evaluated. The PER and NPR values were lower in test groups compared to the control group. TD did not differ between M30 and M50. The animals that were fed the macauba kernel flour had lower concentrations of total cholesterol and triglycerides compared to the control group. The concentration of acetic and propionic acids was higher and the fecal pH was lower in M30 and M50 groups compared to the control group. Gene expression of aminopeptidase (AP) and sodium-glucose transport protein 1 (SGLT1) did not differ among the groups, and the M50 group had lower expression of peptide transporter 1 (PepT1) and sucrase isomaltase (SI) than the control group. Crypts thickness was higher in the M50 group compared to the other groups, while the intestinal muscle layer width did not differ among groups. Therefore, macauba kernel flour proved to be a good plant protein alternative, and its consumption reduced blood lipids and increased short chain fatty acids content.