Abstract Background: Evidence suggests that estrogen plays a preventive role in primary liver cancer development, and it might be thought that isoflavones, which are structurally similar to estrogens and bind to estrogen receptors, are associated with the risk of liver cancer. We investigated this suspected association by measuring plasma concentrations of isoflavones in a nested case–control study of a population-based prospective cohort in Japan. Methods: From 18,628 target participants ages 40 to 69 years who returned the baseline questionnaire and provided blood samples, we selected those with either hepatitis B or hepatitis C virus infection at baseline (n = 1,544). Among these, 90 (28 women and 62 men) were newly diagnosed with primary liver cancer from 1993 through 2006; they were matched with 175 controls (54 women and 121 men). Plasma concentrations of isoflavones (genistein, daidzein, glycitein, and equol) were measured using triple quadrupole tandem liquid chromatography-mass spectrometry. The ORs of liver cancer development based on plasma concentrations were estimated with a conditional logistic regression model. Results: Basically, distributions of plasma isoflavone concentrations did not differ between the cases and controls. No statistically significant associations of genistein, daidzein, glycitein, and equol with primary liver cancer risk were found in either women or men. Conclusions: In middle-aged Japanese women and men with hepatitis virus infection, plasma isoflavones were unassociated with the occurrence of primary liver cancer. Impact: The role of isoflavones in liver carcinogenesis merits further study using both biomarkers and data on dietary intake of isoflavones. Cancer Epidemiol Biomarkers Prev; 24(3); 532–7. ©2014 AACR.