Abstract: Context: There are few studies of 25-hydroxyvitamin D (25(OH)D) concentrations in healthy adults in Brazil. Objective: This work aimed to estimate the prevalence of vitamin D status and its association with lifestyle, sociodemographic, and anthropometric data in 3 regions of Brazil. Methods: A cross-sectional study was conducted among blood donors of both sexes, living in the cities of Salvador, São Paulo, and Curitiba during summer. Blood samples were collected during the procedure. Serum 25(OH)D and parathyroid hormone (PTH) were measured in the same laboratory using chemiluminescence immunoassays. Lifestyle, sociodemographic, and anthropometric data were gathered by an interview with a standardized questionnaire. Vitamin D deficiency and insufficiency was defined as 25(OH)D levels below 20 ng/mL and below 30 ng/mL, respectively. Results: A total of 1004 healthy adults were evaluated with mean levels of 25(OH)D (28.7 ±9.27 ng/mL) and PTH (34.4±15.1 pg/mL). The standardized prevalence of vitamin D deficiency and insufficiency was in the study population 15.3% and 50.9%: in Salvador 12.1% and 47.6%, in São Paulo 20.5%, and 52.4% and in Curitiba 12.7% and 52.1%, (P=.0004). PTH levels were negatively correlated with 25(OH)D levels. Greater body mass index (BMI) and higher latitude were significant predictors of vitamin D deficiency, whereas skin color (White), longer duration of sun exposure, and current use of dietary supplement were protective. Conclusion: This study confirmed the high prevalence of vitamin D deficiency and insufficiency even in the idsummer in a healthy population of Brazil. Vitamin D levels are associated with sun exposure, latitude, BMI, skin color, and use of supplements.