The active metabolite of vitamin D, 1,25(OH)2D3, has many important roles in birds and mammals. In intensive production, timely monitoring of poultry health is important, and hematological and biochemical tests can be extremely important. The aim of this study was to determine the effect of adding 1,25(OH)2D3-glycosides of plant origin to poultry feed on the total number and population of leukocytes and the concentration of calcium, phosphorus and magnesium in the blood. The study was conducted on 10,000 broiler breeder hens of the Ross 308 line. The experimental group of laying hens was fed with 1,25(OH)2D3-glycoside of plant origin, while the control group consumed commercially available food, according to genetic recommendations. Venous blood samples were taken by wing vein puncture in lithium heparin anticoagulant and in gel tubes. Blood was taken 14, 25 and 35 days after the start of adding vitamin D supplement into feed. The total number of leukocytes in the blood samples was determined together with populations of leukocytes differentiated from the blood smear. The absolute number of individual leukocytes’ populations and the ratio of heterophiles to lymphocytes were calculated. Concentrations of calcium, phosphorus and magnesium were determined in serum samples. In this study, the addition of 1,25(OH)2D3-glycosides of plant origin to laying hens led to a decrease in the total number of heterophiles, lymphocytes and eosinophils with an increase in the percentage of lymphocytes and an increase in blood calcium and magnesium levels. Based on the results, we can conclude that supplementation of laying hens with 1,25(OH)2D3-glycoside after 55 weeks of age temporarily reduces the H/L ratio indicating lower sensitivity to stress, and increases serum calcium concentration, which allows the production of better quality egg shells.