Reduced glutathione (GSH) is an abundant tripeptide present in the majority of cell types. GSH is highly reactive and is often conjugated to other molecules, via its sulfhydryl moiety. GSH is synthesized from glutamic acid, cysteine, and glycine via two sequential ATP‑consuming steps, which are catalyzed by glutamate cysteine ligase (GCL) and GSH synthetase (GSS). However, the role of GSH in cancer remains to be elucidated. The present study aimed to determine the levels of GSH and GSH synthetic enzymes in human colorectal cancer. The mRNA and protein expression levels of GSH, the catalytic subunit of GCL (GCLC) and GSS were significantly higher in the following five colon cancer cell lines: Caco‑2, SNU‑407, SNU‑1033, HCT‑116, and HT‑29, as compared with the normal colon cell line, FHC. Similarly, in 9 out of 15 patients with colon cancer, GSH expression levels were higher in tumor tissue, as compared with adjacent normal tissue. In addition, the protein expression levels of GCLC and GSS were higher in the tumor tissue of 8 out of 15, and 10 out of 15 patients with colon cancer respectively, as compared with adjacent normal tissue. Immunohistochemical analyses confirmed that GCLC and GSS were expressed at higher levels in colon cancer tissue, as compared with normal mucosa. Since GSH and GSH metabolizing enzymes are present at elevated levels in colonic tumors, they may serve as clinically useful biomarkers of colon cancer, and/or targets for anti-colon cancer drugs.