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This study aimed to investigate the effects of different concentrations of in ovo injection of 25-hydroxycholecalciferol (25(OH)D3) on early growth, immune and tibia development in Daheng broilers. The results showed that 25(OH)D3 can significantly improve the hatchability of broilers and improve body weight (p < 0.05). The low concentration of 25(OH)D3 (8 μg/mL) not only significantly increased tibial fresh weight, tibial length, tibial diameter, thymus and liver weight and immune organ index (p < 0.05), but also significantly increased the content of bone metabolism-related factors OC and CaBP in serum and the expression levels of OC in the liver (p < 0.05). However, a high concentration of 25(OH)D3 group (16 μg/mL) significantly increased bursa of Fabricius weight and immune organ index (p < 0.05), the content of immune factor IL-2, IFN-γ and TLR15 in serum and the expression level of IFN-γ and TLR15 in the spleen (p < 0.05). Furthermore, the content of ash, calcium and phosphorus in the tibia was negatively correlated with the expression level of OC and positively correlated with the expression level of ALP (p > 0.05). Our data suggested that 25(OH)D3 has positive effects on the early growth, immune, bone development of broiler during the early stage, which is a supplement to improve disease resistance and skeletal development of broilers.