Nanotechnology has recently played a key role in tackling many aquacultures issues. Hence, the present study targets the evaluation of dietary inclusion of nano iron oxide (nFe2O3) on growth performance, hematology, immune-antioxidant responses, ionic regulation and expression of related genes in Nile tilapia (Oreochromis niloticus). Fish were fed supplementary nFe2O3 at rates of zero (control), 0.5, and 1 g/kg diet for 30 days. Obtained data demonstrated that nFe2O3 significantly (P < 0.05) augmented growth performance (final weight and length, body mass gain, specific growth rate, feed conversion ratio, and length gain rate). Hematological picture {RBCs, Hb, MCV, MCH and MCHC, and leukocytes interpretations (WBCs and monocytes)}; and biochemical indexes including (AST and ALT; total protein; and glucose, and cortisol) were significantly (P < 0.05) improved in nFe2O3 supplemented groups. Plasma ionic concentration was also altered with nFe2O3 supplementation, and 1g nFe2O3 revealed the most marked increase in plasma (Na+) potassium (K+) levels. Similarly, IgM, nitrous oxide (NO), and lysozyme activity, plus superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities showed a remarkable improvement in 1g nFe2O3 group compared to the control. Expression of Insulin-Like Growth Factor-1 (IGF-1) and interleukin 1-β (IL-1β) genes were significantly up-regulated in nFe2O3 supplemented groups. Briefly, dietary nFe2O3 inclusion had enhanced properties on growth; hemato-biochemical; immune, antioxidative profiles; and related genes expression of O. niloticus, with a recommended concentration of 1g nFe2O3.