This study investigated the effects of dietary isoleucine (Ile) on growth performance, intestinal expression of amino acid transporters, protein metabolism-related genes and intestinal microbiota in starter phase Chinese yellow-feathered chickens. FemaleXinguangyellow-feathered chickens (n= 1,080, aged 1 d) were randomly distributed to 6 treatments, each with 6 replicates of 30 birds. Chickens were fed diets with 6 levels of total Ile (6.8, 7.6, 8.4, 9.2, 10.0, and 10.8 g/kg) for 30 d. The average daily gain and feed conversion ratio were improved with dietary Ile levels (P< 0.05). Plasma uric acid content and glutamic-oxalacetic transaminase activity were linearly and quadratically decreased with increasing dietary Ile inclusion (P< 0.05). Dietary Ile level had a linear (P< 0.05) or quadratic (P< 0.05) effect on the jejunal expression of ribosomal protein S6 kinase B1 and eukaryotic translation initiation factor 4E binding protein 1. The relative expression of jejunal 20S proteasome subunit C2 and ileal muscle ring finger-containing protein 1 decreased linearly (P< 0.05) and quadratically (P< 0.05) with increasing dietary Ile levels. Dietary Ile level had a linear (P= 0.069) or quadratic (P< 0.05) effect on the gene expression of solute carrier family 15 member 1 in jejunum and solute carrier family 7 member 1 in ileum. In addition, bacterial 16S rDNA full-length sequencing showed that dietary Ile increased the cecal abundances of the Firmicutes phylum, andBlautia, Lactobacillus, andunclassified_Lachnospiraceaegenera, while decreased that of Proteobacteria,Alistipes, andShigella. Dietary Ile levels affected growth performance and modulated gut microbiota in yellow-feathered chickens. The appropriate level of dietary Ile can upregulate the expression of intestinal protein synthesis-related protein kinase genes and concomitantly inhibit the expression of proteolysis-related cathepsin genes.