This experiment evaluated the effects of dietary 25-hydroxyvitamin D3 (25-OH-D3) supplementation in combination with vitamin D3 on broilers from 1 to 32 days of age. Four hundred one-day-old male broiler chicks were randomly allocated to four treatment groups with ten replicates of ten birds each (n=10). Treatment groups included: (1) control containing vitamin D3 at 5000IU/kg feed (125mcg/kg feed); (2) diet with 2500IU/kg (62.5mcg/kg feed) vitamin D3 plus 2500IU/kg (62.5mcg/kg feed) 25-hydroxycholecalciferol; (3) control diet supplemented with 1250IU/kg (31.25mcg/kg feed) 25-OH-D3; and (4) control diet supplemented with 2500IU/kg (62.5mcg/kg feed) 25-OH-D3. Results demonstrated that supplementation with 25-OH-D3 (1250 or 2500 IU/kg) (31.25 or 62.5mcg/kg feed) combined with vitamin D3 (5000IU/kg) (125mcg/kg feed); significantly improved body weight and feed conversion efficiency compared to the control group. Enhanced digestibility of protein, calcium, and phosphorus was also observed. Blood concentrations of calcium, phosphorus, vitamin D, Newcastle Disease (ND) antibody titers, and influenza virus (H9N1) titers were significantly elevated (p<0.05). Hepatic malondialdehyde concentrations decreased while glutathione peroxidase activity increased (p<0.05) with 25-OH-D3 supplementation. Tibial ash, calcium, and phosphorus content were substantially enhanced in supplemented birds. Gene expression analysis revealed increased expression of occludin and CaBP-D28k tight junction proteins (p<0.05) in the jejunum and elevated interleukin-17 cytokine (IL-17) expression in the duodenum, while IL-10 expression was reduced in all treatment groups compared to controls. These findings indicate that 25-OH-D3 supplementation with vitamin D3 enhances broiler performance, antioxidant capacity, immune function, nutrient digestibility, bone mineralization, and calcium metabolism-related gene expression.