The clinical role of vitamin D in sepsis and mortality prediction is controversially discussed. Therefore, we conducted a prospective cohort study on standard care wards, including 461 patients with suspected sepsis fulfilling two or more SIRS criteria. On the first and third day after onset of SIRS symptoms levels of 25(OH)D, 1,25(OH)D and sepsis biomarkers were analysed for their predictive capacity for identifying infection, bacteraemia and an elevated mortality risk. Additionally, several SNPs associated with vitamin D metabolism were evaluated. Bacteraemic patients (28.5%) presented with significantly lower 1,25(OH)D levels than SIRS patients without bacteraemia on the first and third day, while 25(OH)D did not show a predictive capacity. No significant differences of either 1,25(OH)D or 25(OH)D levels were found between SIRS patients with and without infections or between survivors and non-survivors. Sepsis biomarkers, including procalcitonin and CRP, showed a significantly higher discriminatory capacity for these classification tasks. The vitamin D metabolism-related SNPs analysed did not indicate any association with our outcome measures. In conclusion, 1,25(OH)D but not 25(OH)D showed a minor discriminatory value for the prediction of bacteraemia that was inferior to CRP and PCT but both failed to predict sepsis and mortality in a prospective cohort of SIRS patients.