Background: Vitamin D deficiency has been documented to be prevalent, even in low latitude regions; and this may be related to sun exposure behaviors. The aim of the current study was to assess the association between serum 25-hydroxyvitamin D [25(OH)D] concentrations and lifestyle-related factors in a sample of Brazilian women living at latitude 21º 8' S. Methods: A cross-sectional study was undertaken in 101 women aged 35 years or older in July 2019 to assess the association between 25(OH)D concentration and level of exposure to ultraviolet radiation (UVR), smoking habits, alcohol consumption, and physical activity levels. Age, body mass index (BMI), and postmenopausal status were investigated. Findings: According to the slope coefficient for individual daily UVR levels, the concentration of 25(OH)D increased by 5 nmol / L for each extra Standard Erythema Dose of UVR, regardless of age and BMI (p < 0·001). Postmenopausal women had a significantly higher mean concentration of 25(OH)D (p = 0·01), higher UVR exposure (p = 0·01) and lower BMI (p = 0·005) compared with younger women, independent of other confounders including smoking, alcohol, occupation and physical activity. Interpretation: Although postmenopausal women from Brazil had higher mean concentrations of 25(OH)D than younger women, more studies are necessary to understand how sun exposure and lifestyle variables interfere with these levels. These findings have important public health implications since they suggest that vitamin D deficiency in older age is not inevitable. Funding: This study was funded by an award received by Universities Global Partnership Network - UGPN. KVSS and SLO receive scholarship from CAPES, Brazilian Ministry of Education. HR receives a productivity grant from CNPq.