Higher concentration of insulin-like growth factor-1 (IGF-1) increases postmenopausal breast cancer risk, but evidence for insulin and c-peptide is limited. Furthermore, not all studies have accounted for potential confounding by biomarkers from other biological pathways, and not all were restricted to estrogen receptor (ER)-positive breast cancer. This was a case-cohort study of 1,223 postmenopausal women (347 with ER-positive breast cancer) from the Melbourne Collaborative Cohort Study. We measured insulin, c-peptide, IGF-1, insulin-like growth factor binding protein-3, and biomarkers of inflammatory and sex-steroid hormone pathways. Poisson regression with a robust variance estimator was used to estimate risk ratios (RR) and 95% confidence intervals (95% CI) for ER-positive breast cancer per doubling plasma concentration and for quartiles, without and with adjustment for other, potentially confounding biomarkers. ER-positive breast cancer risk was not associated with doubling of insulin (RR = 0.97, 95% CI, 0.82-1.14) or c-peptide (RR = 1.01, 95% CI, 0.80-1.26). Risk seemed to decrease with doubling IGF-1 (RR = 0.80, 95% CI, 0.62-1.03) and insulin-like growth factor binding protein-3 (RR = 0.62, 95% CI, 0.41-0.90). RRs were not meaningfully different when exposures were modeled as quartiles. RRs were less than unity but imprecise after adjustment for inflammatory and sex-steroid hormone biomarkers. Circulating insulin, c-peptide, and IGF-1 were not positively associated with risk of ER-positive breast cancer in this case-cohort analysis of postmenopausal women. Associations between insulin and c-peptide and risk of ER-positive breast cancer in postmenopausal women are likely to be weak.