Fluoride toxicity is a burgeoning problem in worldwide. It is a serious health problem of the population living in high fluoride region. Many experimental and clinical studies have been reported that the fluoride induces deformities in bones and deterioration in skeletal system but exact mechanism of action is still unknown. In the present study, 50 male subject were recruited from the orthopedic OPD at National Institute of Medical Sciences & Research Hospital, Nims University Rajasthan, Jaipur, India. The age matched controls were selected from the area where fluoride content was less than 1.5 ppm. Serum fluoride and 25-Hydroxy vitamin D (25 OHD) were estimated in both subject and controls. Significant (p<0.001) reduction was observed in the 25 OHD while the concentration serum fluoride was found to be significantly (p<0.001) elevated as compared with their respective controls. It is observed that a positive correlation (r=0.738; p<0.001) between 25OHD and serum fluoride. On the basis of results, it may conclude that correlation of the serum fluoride and 25OHD may be used as early marker for the detection of fluorosis. However, a deep study in rodent model may be required to explore the mechanism.