A greater prevalence of dental fluorosis has been reported in higher- versus lower-altitude communities. This may be due to specific changes in the body burden of fluoride which occurs in humans living at high altitude. This study aimed to quantify the effect of altitude on fractional urinary fluoride excretion (FUFE) and total daily fluoride retention (TDFR) in children living at low- er (<78 m) and higher (>1487 m) altitudes in Nepal. Eighty-nine children, aged 4–5 years completed the study: 42 living at lower- and 47 at higher altitude. Total daily fluoride intake (TDFI) was calculated from diet and toothpaste ingestion. Daily urinary fluo- ride (DUFE) excretion was measured by collecting a 24-hour urine sample and total daily fluoride excretion (TDFE) estimated as “DUFE + faecal fluoride excretion”, assuming 10% of TDFI was excreted through faeces. TDFR was estimated by subtracting TDFE from TDFI. FUFE was calculated as the ratio between DUFE and TDFI. A multivariable-adjusted general linear model was used to investigate differences in fluoride intake, excretion and reten- tion between the 2 altitudes. TDFI was significantly greater at low- er- compared with higher altitude (Mean±SD: 0.066 ± 0.029 v 0.045 ± 0.020 mg/kgbw/day (95% CI: +0.009 to –0.030 mg/kgbw/ day; P = 0.001), and FUFE was significantly greater at lower- v higher altitude (Mean±SD: 54 ± 19% v 46 ± 16% (95% CI: +0.626 to +15.23%; P = 0.044). No statistically significant difference was observed for DUFE (adjusted to TDFI) or TDFR between lower- and higher altitude children. Effects of altitude on TDFR were small and non-significant. However, the statistically significant greater FUFE (%) seen at lower altitude suggests greater body re- tention of fluoride at higher altitude which may increase the risk of dental fluorosis.