Introduction: A greater prevalence of dental fluorosis has been reported in higher- versus lower-altitude communities. This study, for the first time, examined several aspects of fluoride metabolism in children, aged 4–5 years, and their parent, living at lower altitude (<78 m) and higher altitude (>1487) areas in Nepal. Methods: The study assessed total daily fluoride intake (TDFI), 24 h urinary fluoride excretion (UFE), and fluoride concentrations of toe- and finger-nail (FC toenail, FC fingernail) in children and parents as well as fluoride concentration of plasma (FC plasma) in parents. Fractional urinary fluoride excretion (FUFE) was calculated as the ratio between UFE and TDFI. FC toenail, FC fingernail and FC plasma were normalised for TDFI by dividing the variables by TDFI and the ratio was reported as the percentage. Results: In total, 89 children and 80 parents took part in the study: 42 children and 41 parents from the lower altitude area; 47 children and 39 parents from the higher altitude area. Fluoride concentration of drinking water was significantly (P < 0.001) higher at lower altitude (0.395 mg F/l) than at higher altitude (0.104 mg F/l). TDFI was significantly (p < 0.001) higher in both children and parents living in lower altitude than those living at higher altitude. There was a statistically significant (p = 0.044) difference in the mean FUFE of children living at lower altitude (53%) and higher altitude (46%). However, no significant difference in FUFE was found between parents living at lower altitude (47%) compared with higher altitude (41%). In both children and parents, no statistically significant differences in normalised FC toenail, FC fingernail were found between the two altitude areas. However, normalised FC plasma was statistically significantly (P = 0.005) higher in parents living at higher altitude (0.15%) compared with those living at lower altitude (0.11%). Conclusion: The results suggest that higher altitude living results in decreased urinary fluoride excretion, and consequently increased fluoride retention in children for a given dose (amount) of fluoride.