BACKGROUND: Recent studies have suggested an association between high dietary intake of calcium and the risk of prostate cancer. Calcium-rich diet has been suggested to affect the serum levels of Vitamin D, and thereby promote cancer. We conducted the largest study of the association between prediagnostic serum levels of calcium and the risk of prostate cancer. OUTLINE: We examined the incidence of prostate cancer in relation to prediagnostic serum calcium levels in a prospective cohort study of 22,391 healthy Swedish men, of which 1,539 incident cases of prostate cancer were diagnosed during the 30 years of follow-up until December 2006. MATERIAL AND METHODS: Serum levels of calcium were measured at baseline, and categorized into quartiles. Cox regression was used to estimate the adjusted hazard ratios (HR) with 95% confidence intervals (CI). RESULTS: We found no evidence of an association between prediagnostic serum levels of calcium and risk of prostate cancer (HR for trend = 0.99 [95% CI;0.94-1.03]). However, a moderate significant negative association was seen in men with a BMI above 25 and aged below 45 years at baseline (Highest vs. lowest quartile, HR = 0.63 [95% CI;0.40-0.99]). CONCLUSION: These data do not support the hypothesis that high serum calcium levels is a risk factor for prostate cancer. On the contrary, the data suggest that high serum levels of calcium in young overweight men may be a marker for a decreased risk of developing prostate cancer.