This study was conducted to determine the effects of supplementation of nanoparticles of zinc oxide in laying hen diets on laying performance, egg quality, nutrient digestibility, and zinc retention. A total of one hundred and twenty Bovans Brown laying hens (55-week-old) were assigned to four treatment diets including nanoparticles of zinc oxide at 0, 20, 40, or 60 mg/kg, respectively, for 12 weeks. Each treatment had six replicates with five hens each. The results revealed that feed conversion ratio was significantly improved (linear, P<0.01) with increasing levels of nanoparticles of zinc oxide in laying hens diet from 55-59, 59-63, 63–67 and 55-67 weeks of age. Hen day egg production, egg mass were significantly increased (P<0.05) by supplementation of nanoparticles of zinc oxide during the experimental periods. Moreover, Haugh unit, shell thickness and eggshell percentage were improved (P<0.01) with increasing levels of nanoparticles of zinc oxide. Digestibility of crude protein, ether extract and crude fiber linearly increased (P<0.001) with increasing levels of supplementation. Interestingly, the serum biochemical analyses revealed that serum cholesterol, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), urea and somewhat creatinine linearly decreased with increasing Nano-ZnO levels in the diets. In conclusion, inclusion of nanoparticles of zinc oxide at 20, 40, or 60 mg/kg had improved productive performance, Haugh unit, shell quality, nutrient digestibility, cholesterol, liver and kidney functions and can be used as an effective feed additive in laying hens diets.