Furan, a possible human carcinogen, is found in heat treated foods and tobacco smoke. Previous studies have shown that humans are capable of converting furan to its reactive metabolite, cis-2-butene-1,4-dial (BDA), and therefore may be susceptible to furan toxicity. Human risk assessment of furan exposure has been stymied because of the lack of mechanism-based exposure biomarkers. Therefore, a sensitive LC-MS/MS assay for six furan metabolites was applied to measure their levels in urine from furan-exposed rodents as well as in human urine from smokers and nonsmokers. The metabolites that result from direct reaction of BDA with lysine (BDA-Nα-acetyllysine) and from cysteine-BDA-lysine cross-links (N-acetylcysteine-BDA-lysine, N-acetylcysteine-BDA-Nα-acetyllysine and their sulfoxides) were targeted in this study. Five of the six metabolites were identified in urine from rodents treated with furan by gavage. BDA-Nα-acetyllysine, N-acetylcysteine-BDA-lysine and its sulfoxide were detected in most human urine samples from three different groups. The levels of N-acetylcysteine-BDA-lysine sulfoxide were more than 10 times higher than the corresponding sulfide in many samples. The amount of this metabolite was higher in smokers relative to non-smokers and was significantly reduced following smoking cessation. Our results indicate a strong relationship between BDA-derived metabolites and smoking. Future studies will determine if levels of these biomarkers are associated with adverse health effects in humans.