Recent studies provide new mechanistic insights that isoleucine (ILE) regulates protein synthesis through genomic approaches. An 8-week feeding trial was performed to determine dietary ILE requirement based on growth performance, body composition, amino acid (AA) retention, and mRNA levels of myogenic regulatory factors (MRFs), including myogenic differentiation (MyoD), myogenin (MyoG), and myostatin (Mst) as negative regulatory of myogenesis, and mammalian target of rapamycin (mTOR) genes in fingerling Nile tilapia, Oreochromis niloticus. Fish (n = 288; 2.46 ± 0.10 g; mean ± SD) were randomly distributed into six treatments with four replicate groups of 12 fish. Fish were fed diets with graded levels of analyzed ILE at 9.08 (ILE1), 12.13 (ILE2), 15.11 (ILE3), 18.02 (ILE4), 21.15(ILE5), and 24.06 g kg−1 diet (ILE6). Fish were hand-fed, six times a day, until apparent satiety. At the end of the feeding trial, incremental levels of dietary ILE significantly affected body weight gain (BWG), feed efficiency ratio (FER), protein retention efficiency (PRE), and essential and non-essential AA retention. Also, the relative expression of MyoD and MyoG genes in the white skeletal muscle and mTOR gene in the liver was up-regulated in fish fed diet ILE3 (P > 0.05). Optimal branched-chain amino acid (BCAA) ratio (Ile:Leu:Val) was found at 1:1.3:0.9. Based on one-slope broken line model of BWG, the optimal dietary ILE requirement for fingerling Nile tilapia was estimated at 13.67 g kg−1. The results indicate that appropriate dietary ILE level at 5.0% dietary protein could improve protein synthesis and positively regulates muscle growth-related and mTOR genes in fast-growing Nile tilapia.