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Background: Bacterial vaginosis (BV), the leading cause of vaginal discharge, is associated with multiple adverse health outcomes; however, its etiology is unknown. BV treatment is not very effective, thus prevention approaches are needed. Studies investigating the impact of vitamin D on the risk of BV have had mixed findings, including two studies reporting increased risk of recurrent BV for women with higher vitamin D. Materials and Methods: Participants were nonpregnant women in a prospective fibroid study of African Americans (ages 23-34 years) from the Detroit area. The exposure was seasonally adjusted annual mean serum 25-hydroxyvitamin D [25(OH)D] at enrollment. The outcome was self-reported doctor-diagnosed BV over approximate to 20 months between baseline and follow-up. Multivariable-adjusted binomial regression models estimated the risk of BV for a doubling of 25(OH)D and sufficient (20ng/mL) versus deficient (<20ng/mL) 25(OH)D. Results: In total, 1459 women were included. Median 25(OH)D was 15.2ng/mL and 73% were deficient. Sixteen percent of participants reported BV diagnoses over follow-up, 78% of whom had recurrent BV. In multivariable-adjusted analyses, a doubling of 25(OH)D was associated with an increased, rather than the hypothesized decreased, risk of self-reported BV (risk ratio [RR] 1.22, 95% confidence interval 1.02-1.48). Sufficient women also had a significantly higher, rather than lower, risk of self-reported BV (RR 1.31). Results were robust to sensitivity analyses, and post hoc analyses showed no evidence of reverse causation. Conclusions: Overall, our findings do not support vitamin D deficiency as a risk factor for BV in these young, nonpregnant African American women.