This study examined the effects of varying dietary sorghum levels on growth, intestinal microbiome, liver function, and muscle quality in juvenile Nile tilapia (Oreochromis niloticus). Six diets, each with 300 g/kg crude protein and 17 MJ/kg gross energy, were formulated by twin-screw extrusion technology. These diets included 0 (control), 40, 80, 120, 160, and 200 g/kg sorghum, replacing wheat flour and wheat bran incrementally. A total of 540 healthy juvenile tilapia (7.86 +/- 0.03 g) were randomly allocated to six groups, with each group consisting of three cage replicates (30 fish per replicate) for a 10-week feeding trial. As dietary sorghum inclusion increased, final body weight, weight gain, and protein efficiency ratio initially rose and then declined, peaking at 40 g/kg sorghum level, but it was not different from the other groups. However, sorghum inclusion significantly altered the beta diversity of intestinal microbiome, notably increasing polysaccharide-catabolizing bacteria at 40 g/kg sorghum. When the inclusion level of sorghum in the diet was increased to 80-200 g/kg, there was a notable decrease in the mRNA expression levels of intestinal pro-inflammatory cytokines, including interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor-alpha-1. At the same time, the mRNA expression level of the intestinal anti-inflammatory cytokine transforming growth factor beta-1 showed an upward trend. The mRNA expression levels of key intestinal tight junction proteins, such as claudin, occludin, and zonula occludens-1, reached their highest point when the sorghum inclusion level was 80 g/kg, after which a downward trend was observed. Similar trends were observed in hepatic phosphofructokinase activity and mRNA expression level. Conversely, hepatic phosphoenolpyruvate carboxykinase activity increased with higher sorghum levels (40-200 g/kg). Hepatic lipid and glycogen contents increased at dietary sorghum levels exceeding 160 g/kg. Improved myofiber development and density were achieved at the 80-120 g/kg dietary sorghum inclusion. In conclusion, optimal physiological outcomes were achieved with 40-80 g/kg dietary sorghum inclusion, without compromising growth performance.