The relationships between bone turnover, the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and vitamin D are complex, but still not fully explained. The GH/IGF-1 axis and vitamin D could modulate their metabolism mutually and influence activation of cell proliferation, maturation, and mineralization as well as bone resorption. The aim of this study was to evaluate the reciprocal associations between bone formation markers [alkaline phosphatase (ALP), bone alkaline phosphatase (BALP)], the GH/IGF-1 axis and 25-hydroxyvitamin D [25(OH)D] in children with growth hormone deficiency at baseline and during recombinant human growth hormone (rhGH) therapy. ALP, BALP, 25(OH)D and IGF-1 concentrations were evaluated in 53 patients included in this prospective three-year study. ALP, BALP and IGF-1 increased during rhGH therapy. Baseline ALP correlated positively with baseline height velocity (HV). ALP and BALP at 12 months correlated positively with HV in the first year of therapy. We found positive correlations between ALP and IGF-1 at baseline and during the first year of therapy, between BALP at 12 months and rhGH dose in the first year of therapy, and between mean doses of cholecalciferol in the first year of rhGH therapy and early changes in BALP during rhGH therapy. Our results indicate that vitamin D supplementation enhances the effect of rhGH on bone formation activity, which could improve the effects of rhGH therapy. ALP and BALP are useful in early prediction of the effects of rhGH therapy, but their utility as long-term predictors is not sufficient.