BACKGROUND: Acrylamide (AA) is classified as "probably carcinogenic to humans (Class 2A)" by the International Agency for Research on Cancer. AA causes cancer owing to its mutagenic and genotoxic metabolite, glycidamide (GA), and its effects on sex hormones. Both AA and GA can interact with hemoglobin to hemoglobin adducts (HbAA and HbGA, respectively), which are considered appropriate biomarkers of internal exposure of AA. However, few epidemiological studies reported an association of HbAA and HbGA with breast cancer (BC). METHODS: We conducted a nested case-control study within the Japan Public Health Center-based Prospective Study cohort (125 cases and 250 controls). Cases and controls were categorized into tertiles (Lowest, Middle, and Highest) using the distribution of HbAA or HbGA levels in the control group and estimated odds ratios (ORs) and 95% confidence intervals (CIs) using conditional logistic regression, adjusting for potential confounders. RESULTS: No association was observed between HbAA (ORHighest vs. Lowest, 1.34, 95% CI, 0.69-2.59), HbGA (ORHighest vs. Lowest, 1.46, 95% CI, 0.79-2.69), their sum HbAA+HbGA (ORHighest vs. Lowest, 1.36, 95% CI, 0.72-2.58) and BC; however, some evidence of positive association was observed between their ratio, HbGA/HbAA, and BC (ORHighest vs. Lowest, 2.19, 95% CI, 1.11-4.31). CONCLUSIONS: There was no association between biomarkers of AA and BC. IMPACT: It is unlikely that AA increases BC risk; however, the association of AA with BC may need to be evaluated, with a focus not only on the absolute amount of HbAA or HbGA but also on HbGA/HbAA and the activity of metabolic genes.