In vitro and in vivo studies have associated iron with both the initiation and promotional stages of carcinogenesis. We investigated whether iron was associated with colorectal cancer in a nested case-control study within the α-tocopherol, β-carotene cancer prevention study cohort. Exposure was assessed at baseline, using a 276-item food frequency questionnaire and a fasting serum sample. The study included 130 colorectal cancer cases (73 colon cancers and 57 rectal cancers) and 260 controls. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Supplemental iron intake was only reported for 4 cases and 18 controls; therefore, we were unable to obtain meaningful results for this variable. Comparing the highest to the lowest quartiles, there was an inverse association between serum ferritin and colorectal cancer risk (OR = 0.4, 95% CI = 0.2–0.9) and a suggestion of an inverse association between dietary iron and colorectal cancer risk (OR = 0.4, 95% CI = 0.1–1.1). In addition, serum ferritin, serum iron and transferrin saturation were all inversely associated with colon cancer risk specifically (OR = 0.2, 95% CI = 0.1–0.7, p trend = 0.02; OR = 0.2, 95% CI = 0.1–0.9, p trend = 0.05; OR = 0.1, 95% CI = 0.02–0.5, p trend = 0.003, respectively), whereas serum unsaturated iron binding capacity was positively associated with colon cancer risk (OR = 4.7, 95% CI = 1.4–15.1, p trend = 0.009). In summary, we found a significant inverse association between several serum iron indices and colon cancer risk. © 2006 Wiley-Liss, Inc.