Dietary protein is one of the most important nutritional factors in aquaculture. The aim of this study was to examine the effects of dietary protein levels on the gut microbiome and the liver and serum levels of metabolites in tilapia. Tilapia were fed a diet with a low (20%), moderate (30%), or high (40%) content of crude protein, and the homeostasis of the gut microbiome and metabolic profile of the liver and serum were analyzed. The results showed no significant differences in the diversity and richness of the gut microbiome among the groups; however, there were differences in the microbial composition of the gut. The metabolome analysis of liver samples revealed a difference in the glucose level among the groups, with the highest glucose level in fish fed a high protein diet. In addition, there were significant differences in the levels of tyrosine, guanosine, and inosine among the metabolome analysis of serum samples of these groups. In summary, diets with different protein levels could affect the composition of gut microbiota and the dynamic balance of microbial communities. Dietary protein content can also affect glycolysis and amino acid metabolism in tilapia.