Background The effects of safflower oil and vitamin C (Vit. C) inclusion in broiler chicken diets on the growth performance, apparent ileal digestibility coefficient “AID%” of amino acids, intestinal histology, behavior, carcass traits, fatty acid composition of the breast muscle, antioxidant and immune status for a 35-day feeding period were evaluated. A total of 300 three-day-old Ross chicks (58.25 g ± 0.19) were randomly allotted in a 2 × 3 factorial design consisting of two levels of vitamin C (0 and 400 mg/kg diet) and three levels of safflower oil (0, 5, and 10 g/kg diet). Results An increase in the final body weight, total body weight gain, total feed intake, and the relative growth rate (P <  0.05) were reported by safflower oil and vitamin C inclusion. Dietary supplementation of safflower oil and vitamin C had a positive effect (P <  0.05) on the ingestive, resting, and feather preening behavior. Vitamin C supplementation increased (P <  0.05) the AID% of lysine, threonine, tryptophan, arginine, and valine. Safflower inclusion (10 g/kg) increased (P <  0.05) the AID% of methionine and isoleucine. Safflower oil inclusion increased (P <  0.05) the levels of stearic acid, linoleic acid, saturated fatty acids, and omega-3 fatty acids (ω-3) in the breast muscle. In contrast, the supplementation of only 10 g of safflower oil/kg diet increased (P = 0.01) the omega-3/omega-6 (ω-3/ω-6) fatty acids ratio. Vit. C supplementation increased (P <  0.05) the CAT serum levels, SOD, and GSH enzymes. Dietary supplementation of safflower oil and vitamin C improved the intestinal histology. They increased the villous height and width, crypt depth, villous height/crypt depth ratio, mucosal thickness, goblet cell count, and intra-epithelium lymphocytic lick cell infiltrations. The serum levels of IgA and complement C3 were increased (P <  0.01) by Vit. C supplementation and prominent in the 400 vit. C +  10 safflower Oil group. Conclusion A dietary combination of safflower oil and vitamin C resulted in improved growth rate, amino acids AID%, intestinal histology, welfare, immune and antioxidant status of birds, and obtaining ω-3 and linoleic acid-enriched breast muscles. The best inclusion level was 400 vit. C +  10 safflower Oil.