A study was carried out to evaluate the effect of in-ovo injection of iron–methionine chelates (Fe-Meth) or iron nano-particles (FeNPs) and post hatch feeding  with diets fortified with folic acid on productive performance and some physiological responses of Ross broiler chickens. A total of 450 fertile eggs were allocated to 5 treatment groups in the incubator, each of which included 3 replicates (30 eggs each). Experimental treatments consisted of a 5 × 2 factorial arrangement design with 5 levels of in-ovo injection solutions at day seven of incubation and two dietary levels of folic acid (FA) (0.0 or 0.5g/kg, diet) after hatching. In-ovo injection treatments were divided into 5 groups: control non- injected (T1), injected with 0.1 ml physiological saline (T2),  sham-injected control (T3),injected with 0.1 ml of 75 ppb of iron nano-particles (T4, FeNPs), while the last group (T5) was injected with 0.1 ml of 75 ppb organic iron (Fe- meth). Results showed that in-ovo injection of different iron sources significantly improved live body weight and weight gain of broiler chicks, and enhanced FCR at 3 and 5 weeks of age compared to other groups. Broilers fed  folic acid fortified diets displayed significantly heavier Live body weight (LBW) and body weight gain (BWG) than the those fed the control un-supplemented diet. The combined interaction effect of in-ovo iron injection and dietary folic acid supplementation showed the best productive performance figures during the whole experimental period. Different iron sources had no significant effect on all plasma protein fraction, Lipids profile except a higher concentration of high density lipoprotein (HDL) in plasma  recorded in-ovo injection of different iron sources compared with different controls. Plasma hemoglobin, globulin fractions(α, β, and γ-globulin), transferrin and Tri-iodothyronine concentration were significantly higher in both injected groups compared to the different control groups. Birds fed FA treatment recorded the lower significant values of plasma cholesterol, triglyceride and LDL, but, the birds fed FA recorded the higher significant value of plasma HDL, plasma hemoglobin, globulin fractions(α, β, and γ-globulin), transferrin and tri-iodothyronine concentration levels compared with birds fed with or without FA. In conclusion, in-ovo injection of Fe-Meth. or FeNPs and fed the diet fortified with FA were sufficient to improve broiler performance or physiological response in broiler chickens.