This study investigated the effects of modulation of the amino acid profile on growth performance and gut health in weaned pigs fed an antibiotic-free, low-protein diet. In experiment 1, 5 treatments were included: a control diet with antibiotics; a low-protein diet with antibiotics; a low-protein diet without antibiotics (LP); a LP diet with 10% more dietary essential amino acids (LP110); and an LP110 diet with 12% more dietary Met + Cys, Thr and Trp. The intestinal digestive enzyme activity and morphology were improved with the increase in dietary essential amino acid levels, while the growth performance was decreased, indicating that the dietary amino acid level was too high. In experiment 2, all 5 treatments of experiment 1 were included, plus a LP diet with 5% more dietary essential amino acids (LP105) and an LP105 diet with 6% more dietary Met + Cys, Thr and Trp. The LP105 treatment showed optimal feed efficiency, a reduced plasma endotoxin concentration, and an increased fecal lactate concentration and increased abundances of Prevotellaceae and Roseburia bacteria. Our results demonstrate that the optimal amino acid profile in an antibiotic-free, low-protein diet can efficiently improve growth performance and gut health and modulate the fecal microbial structure in weaned pigs.