BACKGROUND/OBJECTIVE: Previous cohort studies examining the association of serum antioxidant levels and risk of colorectal cancer have used a single (baseline) measurement only. In the present study, we assessed the association of serum levels of eight antioxidant nutrients in relation to risk of colorectal cancer, using repeated measurements. SUBJECTS/METHODS: Data on a subsample of women in the Women's Health Initiative with repeated measurements of serum retinol, α-carotene, β-carotene, β-cryptoxanthin, lutein+zeaxanthin, lycopene, α-tocopherol and γ-tocopherol during follow-up were included in the analysis. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CIs). RESULTS: Among 5477 women with baseline serum antioxidant values, 88 incident cases of colorectal cancer were identified over a median follow-up time of 12 years. Serum antioxidants measured at baseline generally showed no association with risk of colorectal cancer, although serum β-carotene at baseline showed a non-significant inverse association with colon cancer alone. Furthermore, using the repeated measurements of β-carotene, the average of all measurements was inversely associated with risk of both colorectal and colon cancer: HRs for highest vs lowest tertile 0.54, 95% CI 0.31-0.96, and 0.47, 95% CI 0.25-0.88, respectively. No associations were seen with other antioxidant nutrients in the repeated measure analyses. CONCLUSIONS: In this study, baseline levels of antioxidant nutrients were not associated with risk of colorectal or colon cancer; however, using repeated measures, a relatively high serum level of β-carotene (average of all measurements) was inversely associated with risk of colon and colorectal cancer in postmenopausal women.