Abstract: Background: A large proportion of women living in tropical regions experience vitamin D deficiency, with lifestyle being one of the predisposing factors. Women with single nucleotide polymorphisms of the vitamin D receptor gene living in rural and urban areas have different lifestyles. Objective: This study aimed to determine the differences in serum levels of 25-hydroxyvitamin D [25(OH)D] in women with single nucleotide polymorphisms of the vitamin D receptor gene living in rural and urban areas. Methodology: The present study used a cross-sectional design involving 100 healthy women, who were evaluated for several parameters including serum levels of 25(OH)D, body mass index, body fat, abdominal circumference and lifestyle, which included types of work, duration of exposure to sunlight, sunscreen use, vitamin D intake and physical activity. Statistical analysis included chi-square and independent t-test. Results: From the study population, the prevalence of vitamin D deficiency was 70%, with insufficiency at 29%, sufficiency at 1% and none of the subjects having normal results. Genotype examination showed that homozygote mutant and heterozygote types were found in both groups and only one subject had a homozygote wild type. The average serum level of 25(OH)D was 20.24±4.43 ng mL–1 for the rural group and 14.9±3.64 ng mL–1 for the urban group, with the difference between the two groups significant (p = 0.001). Conclusion: The results show that lifestyle differences between rural and urban areas may affect vitamin D levels given that vitamin D deficiency is still found in both settings.