In a recent study, we demonstrated that the variant allele of rs2480258 within intron VIII of CYP2E1 is associated with reduced levels of mRNA, protein, and enzyme activity. CYP2E1 is the most important enzyme in the metabolism of acrylamide (AA) by operating its oxidation into glycidamide (GA). AA occurs in food, is neurotoxic and classified as a probable human carcinogen. The goal of the present study was to further assess the role of rs2480258 by measuring the rate of AA > GA biotransformation in vivo. In blood samples from a cohort of 120 volunteers, the internal doses of AA and GA were assessed by AA and GA adducts to hemoglobin (Hb) measured by mass spectrometry. The rate of biotransformation was assessed by calculating the GA-Hb/AA-Hb ratio. To maximize the statistical power, 60 TT was compared to 60 CC-homozygotes and the results showed that TT homozygotes had a statistically significant reduced rate of biotransformation. Present results reinforced the notion that T-allele of rs2480258 is a marker of low functional activity of CYP2E1. Moreover, we studied the role of polymorphisms (SNPs) within glutathione-S-transferases (GSTs) enzymes and epoxide hydrolase (EPHX), verifying previous findings that SNPs within GSTs and EPHX influence the metabolism rate.