Colorectal cancer is not strictly considered a tobacco-related malignancy but modest associations have emerged from large meta-analyses. Most studies, however, use self-reported data, which is subject to misclassification. Biomarkers of tobacco exposure may reduce misclassification and provide insight into metabolic variability that potentially influences carcinogenesis. Our aim was to identify metabolites that represent smoking habits and individual variation in tobacco metabolism, and investigate their association with colorectal cancer. In a nested case-control study of 255 colorectal cancers and 254 matched controls identified in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, baseline serum was used to identify metabolites by ultra-high performance liquid-phase chromatography and mass spectrometry, as well as gas chromatography with tandem mass spectrometry. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression. Self-reported current smoking was associated with serum cotinine, O-cresol sulfate, and hydroxycotinine. Self-reported current smoking of any tobacco (OR=1.90, 95% CI:1.02-3.54) and current cigarette smoking (OR=1.51, 95% CI:0.75-3.04) were associated with elevated colorectal cancer risks, although the latter was not statistically significant. Individuals with detectable levels of hydroxycotinine had an increased colorectal cancer risk compared to those with undetectable levels (OR=2.68, 95% CI:1.33-5.40). While those with detectable levels of cotinine had a suggestive elevated risk of this malignancy (OR=1.81, 95% CI:0.98-3.33), those with detectable levels of O-cresol sulfate did not (OR=1.16, 95% CI:0.57-2.37). Biomarkers capturing smoking behavior and metabolic variation exhibit stronger associations with colorectal cancer than self-report, providing additional evidence for a role for tobacco in this malignancy.