Background: Despite increased awareness of the adverse health effects of vitamin D deficiency, only a few studies have evaluated the vitamin D status (25-hydroxyvitamin D [25(OHD)]) of breastfeeding women and up to now, no information exits for German breastfeeding women. Therefore, the aim of study was to determine the vitamin D status of breastfeeding women compared to non-pregnant and non-breastfeeding (NPNB) women. Methods: This cross-sectional study investigated 124 breastfeeding women and 124 age and season matched NPNB women from the German "Vitamin and mineral status among German women" study. The study participants were recruited from April 2013 to March 2015 and did not take vitamin D supplements. Serum 25(OH)D was analyzed by chemiluminescent immunoassay. Results: Vitamin D deficiency (<25.0 nmol/L) was prevalent in 26.6% of the breastfeeding women. The majority of women (49.2%) showed 25(OH)D concentration between 25.0 and 49.9 nmol/L. In multiple binary logistic regression analysis, breastfeeding women had a 4.0-fold higher odds ratio (OR) (95% confidence interval [CI] 1.8, 8.7) for vitamin D deficiency than NPNB women. For breastfeeding women, the risk of vitamin D deficiency was higher in the winter and spring months (OR: 2.6, 95% CI 1.1, 6.3) and increased with lower longitude per one unit (OR 0.7, 95% CI 0.6, 0.9). Conclusion: Breastfeeding women in Germany had a higher risk of deficient vitamin D levels than NPNB women. In further studies, the optimal vitamin D status for breastfeeding women should be investigated and also the required vitamin D doses to ensure this vitamin D status. Trial registration: German Clinical Trial Register (identification number: DRKS00004789 ).