Introduction: The vitamin D effect on orthodontic tooth movement in pregnant women remains unknown. The aim of this study was to investigate the post administration of vitamin D effect on osteoblast numbers and bone alkaline phosphatase (BALP) expression in the tension side in pregnant rats during orthodontic tooth movement. Materials and Methods: This study was an in vivo animal experiment; 28 healthy female Wistar rats (Rattus norvegicus) (16–20 weeks’ old) were divided into four groups, with or without intramuscular administration of vitamin D, which were to be observed after 7 and 14 days. Pregnancy was induced with pregnant mare serum gonadotropin and human chorionic gonadotropin. Nickel–titanium coil springs with 30 g/mm2 of force were connected between the right maxillary incisors and the right maxillary first molar. After 7 and 14 days, the animals were sacrificed. Statistical Analysis Used: Analysis of variance with post hoc test (P 0.05). Results: The highest number of osteoblasts occurred in the C-7 group with mean ± standard deviation of 20.54 ± 8.4. Statistically significant differences were seen in decreased osteoblast number between groups (P = 0.001, P 0.05) in the expression of BALP. Conclusion: The post administration of vitamin D during orthodontic tooth movement in pregnant rats produced no significant enhancement on BALP expression and osteoblast number.