The dietary chromium needs of tilapia have not been thoroughly examined. The present study evaluated the effects of dietary chromic oxide (Cr2O3) on growth performance, feed utilization, hematological parameters, immune response, and gene expression of juvenile Nile tilapia (Oreochromis niloticus) fed semi-purified diets. Six isonitrogenous (33% crude protein), isoenergetic (17.42 MJ Kg− 1), Cr-free diets containing 0 (control), 3, 6, 12, 24, and 60 mg supplemental Cr2O3 kg− 1 were prepared. The diets were administered to triplicate groups of Nile tilapia (average initial weight = 13 g) maintained in 250-L fiberglass tanks within a recirculating indoor system. The feeding regimen consisted of 3% of the fish's live weight, provided twice daily for 60 days. Significant improvements were observed in growth rates, feed utilization efficiency, digestive enzyme activities, blood parameters, liver function enzymes, and immunological responses. The expression of GLUT2 and PI3KR1 genes was upregulated (P < 0.05) with increasing dietary Cr2O3 up to 24 mg kg− 1 diet and leveled off or decreased with a further increase in Cr2O3 supplementation to 60 mg kg− 1. However, the results of quadratic regression analyses indicated that the optimum responses occurred at about 35 mg Cr2O3 kg− 1 diet. The findings suggest that including Cr2O3 in the diet significantly improves fish growth, feed digestion, nutrient absorption, metabolic processes, and innate immune response. A recommended dietary level of approximately 35 mg kg− 1 is proposed to optimize the performance and health status of farmed Nile tilapia.