Insulin and insulin resistance have been hypothesized to increase the risk of breast cancer as insulin increases breast cell proliferation and inhibits sex hormone binding globulin. Although insulin is directly related to body weight, adiposity is inversely associated with breast cancer risk in premenopausal women but directly related to risk in postmenopausal women. To explore the association between insulin and c-peptide levels and breast cancer risk, we conducted a nested case-control study of predominantly premenopausal women within the Nurses' Health Study II cohort. From 1996 to 1999, blood samples were collected from 29,611 participants. A total of 317 cases were diagnosed after blood collection and before June 2003 and matched to 634 controls; 75% of these women were premenopausal at blood collection. Logistic regression models, controlling for breast cancer risk factors, were used to calculate relative risks (RR) and 95% confidence intervals (95% CI). Among women with fasting blood samples (n = 211 cases), insulin was suggestively inversely associated with breast cancer risk (highest versus lowest quartile: RR, 0.5; 95% CI, 0.3-1.0; P(trend) = 0.06). Among all women, c-peptide was not associated with breast cancer risk (highest versus lowest quartile: RR, 1.1; 95% CI, 0.7-1.7; P(trend) = 0.79); results were similar among fasting samples. These associations did not differ by age, body mass index, or waist-to-hip ratio. Overall, higher levels of insulin and c-peptide were not associated with a higher risk of breast cancer among predominantly premenopausal women.