Aim: This study is aimed to determine whether trimethylamine-N-oxide (TMAO) is a predictor of prognosis of patients with colorectal cancer. Methods: Pretreatment TMAO serum levels were determined in 108 patients with colorectal cancer and 30 healthy controls. Results: Median serum TMAO level was significantly higher in colorectal cancer patients than in healthy controls (p < 0.01). No correlation was observed between the disease-free survival and the type of chemotherapy regimen used, tumor location or lymphovascular invasion. Patients with high serum TMAO level had significantly shorter disease-free survival than patients with low serum TMAO level. Multivariate analysis showed that serum TMAO level and distant metastases were independent prognostic factors. Conclusion: Pretreatment serum TMAO level is identified as a new independent prognostic biomarker in patients with colorectal cancer.