The aim of this study was to investigate how dietary supplementation of nanosize zinc oxide affect zinc retention, egg production and eggshell quality, immune response and serum parameters of aged layers. In trial 1, twenty white Leghorn laying hens (68weeks-old) were assigned to the control, ZnO, organic-Zn (Zn-methionine) and nano-Zn (nanosize ZnO) groups. The Zinc was maintained at a 60mg/kg level in the treatment groups’ diet, while the control group’s diet contained 40mg/kg Zn to evaluate the nutrient retention and zinc bioavailability. In trial 2, eighty old white Leghorn laying hens (68weeks-old) were randomly allotted to four dietary treatments (as trial 1) to evaluate the egg production and eggshell quality, immune response and serum parameters. The results of trial 1 indicated that there were no differences in nutrient retention among the groups, but zinc retention was significantly higher in the nano-Zn and organic-Zn groups than that in the control and ZnO groups (P<0.05). Trial 2 results indicated that eggshell thickness was increased in the nano-Zn and organic-Zn groups compared to the control group (P<0.05); immune responses parameters, including: PHA (phytohemagglutinin) skin challenge test result, GRBC (goat red blood cells) antibody titer and IgG levels exhibited no differences among the groups; serum growth hormone concentration and carbonic anhydrase activity was significantly higher in the nano-Zn and organic-Zn groups compare to control group (P<0.05); serum albumin concentration in organic-Zn group was lower than that of the control group (P<0.05). In conclusion, nanosize zinc for dietary supplementation can increase zinc retention, as well as enhance eggshell thickness, serum carbonic anhydrase activity and growth hormone level of layers. We therefore concluded that nanosized zinc oxide can enhance zinc absorption in the intestine of layers compare to regular zinc oxide.