The levels of hemoglobin adducts of acrylamide (AA–Hb), a biomarker of acrylamide exposure, have not been reported for Japanese subjects. Herein, we determined the AA–Hb levels in a Japanese population and compared them with the estimated dietary intake from the duplicate diet method (DM) and a food frequency questionnaire (FFQ). One-day DM samples, FFQ, and blood samples were collected from 89 participants and analyzed for acrylamide. AA–Hb was analyzed using liquid chromatography tandem mass spectrometry and the N-alkyl Edman method. Participants were divided into tertiles of estimated acrylamide intake and geometric means (GMs) of AA–Hb adjusted for sex and smoking status. A stratified analysis according to smoking status was also performed. The average AA–Hb levels for all participants, never, past, and current smokers were 46, 38, 65, and 86 pmol/g Hb, respectively. GMs of AA–Hb levels in all participants were significantly associated with tertiles of estimated acrylamide intake from DM (p for trend = 0.02) and FFQ (p for trend = 0.04), although no association with smokers was observed. AA–Hb levels reflected smoking status, which were similar to values reported in Western populations, and they were associated with estimated dietary intake of acrylamide when adjusted for sex and smoking status.