We aimed to validate a method for assessing trans-fatty acid (TFA) intake in the Japanese population using the Food Frequency Questionnaire (FFQ) developed in the 1990s from a prospective study that was based on the Japan Public Health Center-based Prospective Cohort Study. For FFQ validation, we included 565 participants (Cohort I: n=215, Cohort II: n=350) aged 40-69 years. We used a 28-day dietary record (DR) over one year and two FFQs administered before and after DR assessment. We calculated total TFA intake, TFA from industrial oils (i-TFA), and TFA from ruminants (r-TFA) considering a database of measurements obtained mainly from Japan. Spearman's rank correlation coefficients (CC) were computed for validity and reproducibility. Energy adjustments were applied using two methods considering the TFA measurement: density method for TFA% of total energy and residual method for TFA g/day. The total TFA intake (% of the total energy intake) was 0.08-0.76% (median, 0.27-0.37%) in DRs of both cohorts, and was 0.00-1.13% (median, 0.30-0.40%) in FFQs. The i-TFA accounted for approximately 50% of the total TFA intake in DRs and approximately 40% in FFQs. For total TFA(% of the total energy intake), CCs were 0.54-0.69, and weighted kappa coefficients were 0.88-0.92 for both cohorts. The de-attenuated CC was 0.46-0.62 for i-TFA (g/day) and 0.57-0.68 for r-TFA (g/day). Our study showed that the validity and reproducibility of TFA intake estimation using the FFQ were reasonable, suggesting its suitability among the Japanese population with low TFA intake.