There are few published studies on the association between vitamin D concentrations and preterm birth (PB) in sub-Saharan Africa. The current study aimed to assess the association between 25-hydroxyvitamin D (25[OH)] D) levels and PB. A matched case-control study (60 women in each arm) was conducted in Medani maternity hospital in central Sudan. The cases were women with spontaneous PB, and healthy women with term deliveries were the controls. The clinical/medical and obstetric history was gathered using a questionnaire. The enzyme-linked immunosorbent assay was used to measure the serum 25(OH)D levels. Women with PB had significantly lower median (interquartile range) 25(OH)D concentrations compared with the controls (18.4 (7.3) ng/mL vs. 20.2 (16.5) ng/mL, = 0.001). Forty-two (70.0%) women with PB and 29 (48.3%) women in the control group had vitamin D deficiency (25(OH)D level ≤ 20 ng/mL). The results of the multivariable logistic regression showed that the 25(OH)D concentrations were negatively associated with PB (adjusted odds ratio (aOR) = 0.92, 95% confidence interval (CI) = 0.87-0.97). Vitamin D-deficient pregnant women were at a higher risk of PB (aOR = 2.69, 95% CI = 1.17-6.23). Low 25(OH)D concentrations were found at the time the variable was determined in women with spontaneous PB and were an independent risk factor for PB.