The effects of vitamin D3 on bone metabolism are complicated. There is a limited study of vitamin D3 on chicken osteoblasts differentiation and mineralization. Furthermore, the application of vitamin D3 metabolites, such as 25-hydroxyvitamin D3 on laying hens has shown various effects on egg production and bone quality. Seldom studies have focused on the long-term supplementation of 25-hydroxyvitamin D3 on laying hen. At last, the unique characteristics of laying hen bone make it an attractive research model to understand further laying hen bone physiology as well as to develop a new strategy to alleviate osteoporosis. In this case, the serial studies were conducted to understand the vitamin D3 molecular actions on chicken osteoblasts, the long-term supplementation of 25-hydroxyvitamin D3 on layer bone development and laying performance, as well as modeling layer bone development pattern. In the first study, 1,25-hydroxyvitamin D3 at various concentrations were subjected to treatment with chicken osteoblasts to understand its role in osteoblast differentiation and mineralization. The results showed the role of vitamin D3 on osteoblasts has a close relationship with cell differentiation. It mainly acted as an inhibitor on cell differentiation and mineralization during the early stage, but a promoter during the late stage. Furthermore, a study exploring the effects of long-term supplementation of 25-hydroxyvitamin D3 in diets on layer bone development and egg laying performance were performed. The results showed early (0-60wk) supplementation of 25-hydroxyvitamin D3 in the diets could increase bone structure size, subsequently, benefit the bone health. Moreover, 25-hydroxyvitamin D3 did not have major effects on eggshell quality, but it increased the egg production up to 60wk. The research results highlight the importance of early and long-term supplementation of 25-hydroxyvitamin D3 on bone development and egg production. At last, a bone modeling trial was conducted to understand the layer bone development pattern. The data showed there were dramatic changes in bone development pattern at the beginning of laying, evidenced by the significant increases in bone volume, bone mineral density, and bone mineral content. This indicates the layer nutrition at the point of producing the first egg may be critical for egg production and skeletal integrity during the later laying period.