The aim of this study was to examine the effect of vitamin D-3 and/or zeolite supplementation in presence of phytase enzyme on serum total Calcium (Ca), inorganic Phosphorus (Pi) levels and Alkaline Phosphatase (ALP) activity in laying hens. A total of 120, 28 weeks old laying hens were used for the study. The laying hens were separated to 4 equal groups (5 replicates). The treatment groups were as follows: control diet (300 FTU phytase per kilogram), trial 1 diet (300 FTU phytase + 400 IU vitamin D-3), trial 2 diet (300 FTU phytase + 400 IU vitamin D-3 + 2% zeolite) and trial 3 diet (300 FTU phytase + 2% zeolite). The experimental period was 16 week. Blood samples were taken on weeks 4, 8, 12 and 16. There were no significant differences between groups for serum ALP activity and Pi levels on weeks 4, 8, 12 and 16. During the study, serum Ca levels were significantly higher in the trial 2 than those of other groups (p<0.05). In conclusion, a significant increase or decrease was not seen in the effect of phytase as a result of the addition of vitamin D-3 or zeolite, respectively. There was a significant phytase, vitamin D-3 and zeolite interaction for serum Ca level.