Three experiments were conducted to study the effects of 25-hydroxycholecalciferol supplementation on BW gain, IL-1β, and 1α-hydroxylase mRNA expression in different organs of broiler chickens following a lipopolysaccharide (LPS) injection. In experiment I, birds were fed a basal diet supplemented with either cholecalciferol (3,000 IU/kg) or 25-hydroxycholecalciferol (69 µg/kg). At 21 and 35 d of age, birds were injected with LPS. Post-LPS injection, birds supplemented with 25-hydroxycholecalciferol gained approximately 2.5% (P = 0.03) and 3.8% (P < 0.01), respectively, more BW than the birds supplemented with cholecalciferol over the 24-h period. In experiment II, birds were fed basal diets supplemented with 25-hydroxycholecalciferol at 6.25, 25, and 50 µg/kg of feed or cholecalciferol at 250 IU/kg of feed. At 35 d of age, birds were injected with LPS. Birds fed 25-hydroxycholecalciferol at 25 and 50 µg/kg and injected with LPS had approximately 7-fold and 3-fold less (P = 0.010) IL-1β mRNA in the liver compared with those birds fed 6.25 µg/kg of 25-hydroxycholecalciferol and the cholecalciferol (250 IU/kg) group. In experiment III, birds were fed a basal diet supplemented with either cholecalciferol (3,000 IU/kg) or 25-hydroxycholecalciferol (69 µg/kg). At 28 d of age, birds were fed 25-hydroxycholecalciferol and injected with LPS had 1.1-fold less (P < 0.01) IL-1β mRNA in the liver than the cholecalciferol-fed group. After an LPS injection, birds supplemented with 25-hydroxycholecalciferol had increased 1α-hydroxylase mRNA amounts in the liver (P = 0.07). In conclusion, 25-hydroxycholecalciferol supplementation at higher doses improved growth performance and decreased inflammatory gene IL-1β mRNA amounts in the liver post-LPS injection.