Background: 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. Methods: 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. Results: 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). Conclusion: 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.