Objective:The aim of this study was to assess urine fluoride concentration, nutritional status, and dental fluorosis in adolescent students living in the rural areas of Guanajuato, Mexico.Materials and Methods:A cross-sectional study was conducted including participants aged 11–20 years. The presence and severity of dental fluorosis was registered according to the Thylstrup and Fejerskov index (TFI) criteria. Anthropometric measures were also recorded. Urine sample of the first morning spot was recollected to assess urine fluoride concentration by using the potentiometric method with an ion-selective electrode. Water samples were also recollected and analyzed. Bivariate tests were performed to compare urine fluoride concentration according to different variables such as sex, body mass index, and TFI. Nonparametric tests were used. A logistic regression model was performed (SPSS® 21.0).Results:This study included 307 participants with a mean age of 15.6 ± 1.6; 62.5% of the participants showed normal weight. A total of 91.9% of the participants had dental fluorosis, and 61.6% had TFI > 4. Mean fluoride content in urine ranged between 0.5 and 6.65 mg/L, with a mean of 1.27 ± 1.2 mg/L. Underweight children showed greater urine fluoride concentration. The increment of urine fluoride was a related (OR = 1.40) to having severe dental fluorosis.Conclusions:Most of the studied population had moderate or severe dental fluorosis. Urine fluoride concentration was related to fluorosis severity and nutritional status. Underweight children showed greater urine fluoride concentration as well as severe dental fluorosis.