In poultry production, vaccination is an effective measure to protect chickens from diseases. Vaccination, however, is a stressor that may induce stress responses that interfere with the growth and development of chickens. The interaction between the skeletal and immune systems on bone quality has gained more attention. In the present study, the influence of high frequency vaccinations on the bone development of layer pullets was investigated. Thirty 35-day-old SPF White Leghorn layer pullets were obtained and randomly subjected to the following treatments: vaccinated against Newcastle disease (ND) with LoSota vaccine once at 35-day-old (V1, control); 4 times at 35, 49, 63, and 77 d of age (V4); and 7 times at 35, 42, 49, 56, 63, 70, and 77 d of age (V7). The body weight and organ index of the spleen, thymus, and tibia were recorded. The antibody titer and serum and the tibia calcium and phosphorus concentrations were measured. The transcription levels of the IL-6, IL-17, TNF-α, receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) genes were determined in spleen, thymus, and the tibia. The results showed that V7 decreased body weight and increased the ND antibody titer, compared to V1-chickens. The expression levels of IL-6, IL-17, and TNF-α were upregulated in spleen, thymus, and the tibia of V7 chickens. In the tibia, RANKL was upregulated, while OPG was downregulated by V7 treatment. The results indicate that high frequency vaccination induces immune stress and impairs bone development. The results suggest that the augmented cytokine expression in immune organs and the tibia is associated with activation of the OPG/RANKL pathway, which, in turn, enhances osteoclastogenesis. The appropriate frequency of vaccination should support optimal bone development and full immunoprotection in layer pullets.