Background: Whether dark skin produces less vitamin D after UVB radiation than fair skin remains controversial. Objective: To compare 25-hydroxyvitamin D [25-(OH)-D] levels after a single UVB exposure in fair (phototype II-III) and black-skinned (phototype VI) volunteers. Methods: Fair-skinned volunteers (n = 20, 4 males/16 females, mean age: 23.2 years) and black-skinned (n = 11, 6 males/5 females, mean age: 23.8 years) received a single total body UVB exposure (0.022 J/cm2). The 25-(OH)-D levels were measured on days 0, 2 and 6. Results: On day 0, all volunteers were severely vitamin D deficient. On day 2, 25-(OH)-D levels of fair-skinned volunteers increased significantly (median: 11.9-13.3 ng/ml, p < 0.0001), but not in black-skinned people (median: 8.60-8.55 ng/ml, p = 0.843). Again, on day 6, 25-(OH)-D levels of fair-skinned volunteers increased significantly (median: 11.9-14.3 ng/ml, p < 0.0001), but not in black-skinned people (median: 8.60-9.57 ng/ml, p = 0.375). Conclusion: This study suggests that skin pigmentation negatively influences vitamin D synthesis.