In total, 864 fertile eggs from Hubbard breeder hens were obtained from a commercial hatchery and were randomly distributed in a factorial arrangement of 3*3, factorial design with three levels of L-Arginine (L-Arg) (0%, 1% and 2%) and three levels of fresh Royal Jelly (RJ) (0%, 10% and 20%), in nine treatments containing (96 eggs each).  Results showed that in ovo injection with 1, 2% L-Arg, or 10% RJ improved hatchability by 6.25, 7.29 and 8.33%, respectively, compared with those of non-injected treatment. Whereas, injection of 20% RJ insignificantly decreased hatchability percentage compared with non-injected group. The in ovo injection of eggs with 1, 2% L-Arg, or 10 and 20% RJ improved chicks' weight at hatch by 2.34, 1.63, 1.65 and 3.91%, respectively compared with those of non-injected group. Also, injection of eggs by 1, 2% L-Arg or 10 and 20% RJ produced chicks have higher relative intestine weight by 0.3, 1.34, 0.64 and 0.16%, respectively over those of non-injected group.  However, chicks injected with the mixture of 2% L-Arg and 10% RJ recorded reduction in the relative intestine weight (-0.28%) among the other mixtures, compared with the results of non-injected group. Results indicated that the in ovo injection of L-Arg and RJ was effective in increasing the liver glycogen content of Hubbard chicks at hatch, and this trend increase with the increase of level of nutrient and with mixing between both nutrients. Results showed that the treatment levels whatever L-Arg or RJ has insignificant influence on villi width, length and surface area. Furthermore, the injection of both 1 and 2% of L-Arg were more effective than other levels 10 and 20% or RJ levels with respect of villi width. Results indicated that the highest relative sodium glucose co-transporter 1 gene expression value produced with Hubbard broiler breeder eggs injected with the studied low levels of L-Arg and RJ. In conclusion, it can be concluded that the in ovo injection with 10% RJ and 2% Arg   may be recommend to apply with Hubbard breeder eggs to achieve good hatching traits. Also, the in ovo injection with 1% Arg+20 % RJ mixture has good improvement on intestine weight and villi measurements (gut development) at hatch.