Although current evidence emphasizes a high prevalence of vitamin D deficiency and an inverse association between serum 25-hydroxyvitamin D (25-OHD) concentration and obesity, no studies have been conducted in Mexican children. The objective was to determine the prevalence of vitamin D deficiency and its association with obesity and lifestyle factors in a sample of school-aged Mexican children. A cross-sectional study of 99 obese and 99 nonobese 6–12 year-old children, skin phototypes III–V, from six public schools was conducted during summer at latitude 25°40', in northeastern Mexico. Anthropometric measurements were determined. Serum 25-OHD was measured by immunoluminometric direct assay. Consumption of foods rich in vitamin D, sunscreen use and vitamin consumption were assessed through applied questionnaires. 62.1% of the subjects had insufficiency of 25-OHD (21–29 ng/ml) and 20.2% had deficiency (<20 ng/ml). Obese subjects (BMI ≥95th percentile for age and gender) had significantly lower concentration of 25-OHD than nonobese. Predictors of 25-OHD concentration were, in order of significance: percentage of body fat, BMI, triceps skin fold, and waist circumference (WC). A significantly higher rate of 25-OHD deficiency was observed in children with inadequate milk/yoghurt consumption, but no difference was found for other foods, physical activity (PA) or screen-time. In a multivariate model, being obese was significantly associated with the risk of 25-OHD deficiency, after adjustment for PA, screen-time, skin phototype, ingestion of milk/yoghurt, fish, cheese, and carbonated beverages. A high prevalence of vitamin D deficiency and an inverse association between serum 25-OHD concentration and obesity was found.