Background: It has been shown that zinc has an effect on physiological responses in animals and birds. On the other hand, dietary phytase in poultry results in increased availability of zinc. OBJECTIVES: This study was conducted to investigate the effects of zinc oxide (ZnO) and Escherichia coli-derived 6-phytase supplemented diets on the plasma metabolites and enzyme activities of broiler breeder hens from 60 to 72 weeks of age. METHODS: A total of 128 breeder hens were randomly assigned to eight dietary treatments, with four replicates of four hens each. Blood concentration of Zn, Ca, P, total protein, cholesterol, triglyceride (TG) and high density lipoprotein (HDL), and plasma activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) were measured. RESULTS: Results showed that supplementary ZnO increased plasma Zn, Ca, P, HDL, and total protein (p<0.01) concentrations, as well as enzyme activities of LDH, ALT and AST (p<0.01). Also, a ZnO-supplemented diet resulted in a decrease in plasma cholesterol and TG (p<0.01) levels. Adding phytase to the diet increased plasma (P) and HDL contents (p<0.01). The interactive effect of phytase × ZnO × period on the plasma levels of Zn, P, total protein, HDL, total cholesterol, and the enzymatic activity of LDH, ALT and ALP was significant. CONCLUSIONS: It is concluded that supplementary ZnO and phytase may improve metabolism and enzymatic activity of aged broiler breeder hens.