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Insulin resistance has been suggested to be associated with an increased risk of breast cancer. Insulin sensitivity can be measured using blood C-peptide, a marker of insulin secretion. It is thus conceivable that blood C-peptide levels may be associated with breast cancer risk. To evaluate this hypothesis, we analyzed data from a subset (143 case-control pairs matched by age and status of menopause) of women who participated in the Shanghai Breast Cancer Study, a population-based, case-control study conducted in Shanghai during 1996-1998. Fasting blood samples were collected from study subjects to measure C-peptide levels. For cancer patients, the samples were collected before any cancer therapy. Conditional logistic regression was used to estimate adjusted odds ratios and 95% confidence intervals related to C-peptide levels. Breast cancer risk was increased with increasing levels of C-peptide (trend test, P = 0.01), with an odds ratio of 2.7 (95% confidence interval = 1.2-5.9) observed for the highest compared with the lowest tertile of C-peptide concentration after adjusting for body mass index and age at the first live birth. The risk was not altered after fully adjusting for other traditional risk factors for breast cancer. This positive association was observed in both pre and postmenopausal women and regardless of the levels of waist-to-hip ratio or body mass index. The results from this study were consistent with the insulin-resistance hypothesis for breast cancer and suggest that increased levels of C-peptide may contribute to the development of breast cancer.