The influence of dietary boron (B) supplementation on some serum parameters and egg-yolk cholesterol was studied in laying hens. A total of 224 eighteen-week-old hens of the Hyline Brown 98 strain were assigned to 7 groups with 4 replicates of 8 hens each after 10 days of adaptation, and they were fed commercial diets supplemented with 0, 5, 10, 50, 100, 200 or 400 mg/kg (diet) B (H3BO3) for 8 weeks. Serum gamma-glutamyl transpeptidase (GGT) activity, albumin, glucose, total cholesterol, HDL- and LDL-cholesterol levels were decreased with all B levels. Except in the group receiving 5 mg/kg B supplementation, decreases were found in serum triglycerides in all groups. Serum aspartate aminotransferase (AST) activity was decreased in the groups receiving 100 mg/kg or higher levels of B. All levels of B supplementation increased lactate dehydrogenase (LDH) activity at weeks 21 and 22, while 10 mg/kg or higher levels of B increased serum globulin, urea and egg-yolk cholesterol levels. The results demonstrate that B supplementation at levels exceeding 5 mg/kg affects serum biochemical parameters and increases egg-yolk cholesterol in laying hens.