To investigate the association of serum 25-hydroxyvitamin D (25(OH)D3) with survival in a large prospective cohort study of colorectal cancer (CRC) patients. The study population consisted of 2,910 patients diagnosed with CRC between 2003 and 2010 who participated in the DACHS study, a multicenter study from Germany with comprehensive long-term follow-up. 25(OH)D3 was determined in serum samples collected shortly after cancer diagnosis by High Performance Liquid Chromatography-Electro Spray Ionization-Mass Spectrometry. Analyses of survival outcomes were performed using Cox regression with comprehensive adjustment for relevant confounders. The majority (59%) of CRC patients were vitamin D deficient (serum 25(OH)D3 levels <30nmol/L). During a median follow-up of 4.8years, 787 deaths occurred, 573 of which were due to CRC. Compared to patients in the highest 25(OH)D3 quintile (>45.20nmol/L), those in the lowest 25(OH)D3 quintile (<11.83nmol/L) had a strongly increased mortality. Adjusted hazard ratios (95%Confidence Interval) were 1.78 (1.39-2.27), 1.65 (1.24-2.21), 1.32 (1.03-1.71) and 1.48 (1.18-1.85) for all-cause mortality, CRC-specific mortality, recurrence-free and disease-free survival, respectively. Subgroup analyses did not show any significant effect modification across strata defined by sex, age, stage, body mass index, or the late entry. Dose-response analyses showed a strong inverse relationship between serum 25(OH)D3 levels and survival endpoints at 25(OH)D3 levels <30nmol/L, and no association with mortality at higher 25(OH)D3 levels. Vitamin D deficiency is highly prevalent in CRC patients and a strong independent predictor of poor prognosis. The possibility of enhancing CRC prognosis by vitamin D supplementation, ideally combined with outdoor physical activity, should be evaluated by randomized controlled trials focusing on patients with vitamin D deficiency.