<b><i>Background:</i></b> Many studies have examined the relationship between vitamin D and specific types of cancer with inconsistent results. Furthermore, to date, no observational studies have demonstrated a clear relationship between vitamin D and total cancer risk. <b><i>Methods:</i></b> We analyzed data from a population-based prospective cohort study including 2,003 initially cancer-free participants from the KORA F4 study with baseline serum 25-hydroxyvitamin D (25(OH)D) measurements (surveyed between 2006 and 2008). We used Cox proportional hazard models to assess the association between 25(OH)D levels and incident cancer risk. <b><i>Results:</i></b> Within a follow-up period of 7 years, 69 of the participants developed cancer. Overall, we observed no significant relationship between serum 25(OH)D levels and cancer risk. The hazard ratio (HR) per 1 ng/ml increase in 25 (OH)D for this relationship was 1.00 (95% confidence interval (CI) 0.97-1.04) adjusting for age, sex, body mass index, and season of blood draw. This was also true in subgroup analysis for prostate cancer (HR 0.95, 95% CI 0.88-1.03), breast cancer (HR 1.03, 95% CI 0.97-1.09), and colorectal cancer (HR 0.97, 95% CI 0.88-1.07). <b><i>Conclusion:</i></b> Our study found no protective effect of 25(OH)D against developing cancer. However, studies with more participants and additional measurements of 25(OH)D are still needed to accurately clarify the relationship between 25(OH)D and total cancer risk.