The aim of this study was to evaluate the effects of in ovo injection of zinc oxide nanoparticles on the hatchability, production performance and immune responses of broilers. A total of 192 fertile eggs obtained from a Ross 308 broiler breeder flock were used. The eggs were divided into 4 experimental groups including a positive control group with normal saline injection, and injection of 50, 75 and 100 ppm nano-ZnO on the first day of incubation period in the air cell and were placed in the incubator. The hatched chicks were allocated to a completely randomized design with 4 treatments and 4 replicates for a 42-day experiment. In ovo injection of nano-ZnO increased hatchability of eggs compared to the control group (P<0.05). The daily feed intake during the starter phase significantly increased in the birds in 50 and 75 ppm nano-ZnO groups (P<0.05). At the grower phase in rearing period the growth rate and feed intake of all the nano-ZnO injected groups were higher than the control group (P<0.05). There was no significant difference in the feed conversion ratio between different treatment groups (P>0.05). The results of antibody titer against the influenza virus at 10 days of age did not differ between the experimental treatments (P>0.05). The heterophil, lymphocytes, monocytes and hematocrit number in the blood of broiler chickens at 10 days of age did not show any significant difference (P>0.05). The results of the present study suggest that in ovo injection of nano ZnO particles had positive effects on early embryo mortality rate, total white blood cells, however the performance traits of the hatched chicks were not significantly affected through in ovo injection of nano ZnO particles.