Abstract Background Glutamine is the most abundant amino acid in the body and plays a vital role in colorectal cancer (CRC) cell metabolism. However, limited studies have investigated the clinical and prognostic significance of preoperative serum glutamine levels in patients with colorectal cancer, and the underlying mechanism has not been explored. Methods A total of 121 newly diagnosed CRC patients between 2012 and 2016 were enrolled in this study. Serum glutamine levels were detected, and their associations with clinicopathological characteristics, systemic inflammation markers, carcinoembryonic antigen (CEA) and prognosis were analysed. In addition, the effect of glutamine depletion on recurrence and metastasis was examined in SW480 and DLD1 human CRC cell lines, and epithelial–mesenchymal transition (EMT)-related markers were detected to reveal the possible mechanism. Results A decreased preoperative serum level of glutamine was associated with a higher T-class and lymph node metastasis (P