Background Vitamin D is critical for bone physiology. In this study, we quantified Vitamin D Binding Protein (VitDBP) levels in saliva as a measure of Vitamin D during orthodontic tooth movement. Methods In this longitudinal study, saliva samples were collected from 73 orthodontic patients for 4 timepoints for the first six months of orthodontic treatment, along with dental casts at the beginning and the end of the study period. The saliva was measured for VitDBP as a biological marker for bone apposition and clinical tooth movement. We used the absolute change in Little's Irregularity Index as a quantitative measure for alignment. In addition, we measured the levels of alkaline phosphatase (ALP) in saliva as a marker of bone turnover. Results Both low (< 2.75 ng/ml) and high (> 6.48 ng/ml) VitDBP levels were associated with reduced tooth movement. Significant (p < 0.05) seasonal changes in VitDBP using a two-season year model were found with lower levels observed in the summer (Apr–Sept) than in the winter (Oct–Mar). Conclusions Clinically significant orthodontic tooth movement is associated with an optimal range of VitDBP in saliva. Normal levels of VitDBP correlated with more orthodontic tooth movement, suggesting a "normal" range of salivary content of VitDBP. Given the strong trend that is independent of the confounding factors (ex. age, race or gender), the predictive value or salivary VitDBP for tooth movement should be studied in larger cohorts in future studies.