AbstractDysregulation of tryptophan metabolism has been linked to colorectal tumorigenesis; however, epidemiological studies investigating tryptophan metabolites in relation to colorectal cancer risk are limited. We studied associations of plasma tryptophan, serotonin and kynurenine with colon cancer risk in two studies with cancer patients and controls, and in one prospective cohort: ColoCare Study (110 patients/153 controls), the Colorectal Cancer Study of Austria (CORSA; 46 patients/390 controls) and the European Prospective Investigation into Cancer and Nutrition (EPIC; 456 matched case‐control pairs). Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for colon cancer risk. Tryptophan was inversely associated with colon cancer risk in ColoCare (OR per 1‐SD = 0.44; 95% CI, 0.31‐0.64) and EPIC (OR per 1‐SD = 0.86; 95% CI, 0.74‐0.99). Comparing detectable vs nondetectable levels, serotonin was positively associated with colon cancer in CORSA (OR = 6.39; 95% CI, 3.61‐11.3) and EPIC (OR = 2.03; 95% CI, 1.20‐3.40). Kynurenine was inversely associated with colon cancer in ColoCare (OR per 1‐SD = 0.74; 95% CI, 0.55‐0.98), positively associated in CORSA (OR per 1‐SD = 1.79; 95% CI, 1.27‐2.52), while no association was observed in EPIC. The kynurenine‐to‐tryptophan ratio was positively associated with colon cancer in ColoCare (OR per 1‐SD = 1.38; 95% CI, 1.03‐1.84) and CORSA (OR per 1‐SD = 1.44; 95% CI, 1.06‐1.96), but not in EPIC. These results suggest that higher plasma tryptophan may be associated with lower colon cancer risk, while increased serotonin may be associated with a higher risk of colon cancer. The kynurenine‐to‐tryptophan ratio may also reflect altered tryptophan catabolism during colon cancer development.