BACKGROUND: Cancer cells undergo a metabolic shift termed the Warburg effect, transitioning from oxidative phosphorylation to aerobic glycolysis and promoting rapid tumor proliferation. Quinone oxidoreductase (NQO1), a cytosolic flavoprotein, is important for reprogramming cancer cell metabolism. Therefore, NQO1's function in aerobic glycolysis and impact on colorectal cancer (CRC) development and progression was investigated. METHODS: The clinical significance of NQO1 was evaluated by analyzing online databases and was substantiated in CRC specimens. NQO1's influence on proliferation, epithelial-mesenchymal transition (EMT), metastasis, apoptosis, and glycolytic pathways in CRC cells was evaluated using in vitro and in vivo experiments. The molecular interactions between NQO1 and microRNA-485-5p (miR-485-5p) were ascertained via quantitative reverse transcription PCR and dual luciferase reporter assays. The molecular mechanisms underlying the miR-485-5p/NQO1 axis and its effects on progression of malignancy and aerobic glycolysis in CRC cell lines were investigated. RESULTS: NQO1 promoted CRC cell proliferation and EMT, augmented their metastatic potential, and suppressed their apoptosis. The NQO1 overexpression-mediated enhancement of glycolytic activity is implicated in the increased proliferation, EMT, and metastatic abilities of, and reduced apoptosis in, CRC cells. Further, miR-485-5p may inhibit the proliferative and invasive traits of CRC cells by directly targeting the 3' untranslated region of NQO1 mRNA. CONCLUSIONS: miR-485-5p/NQO1 signaling axis orchestrates aerobic glycolysis, thereby modulating CRC cell proliferation, metastasis, and apoptosis. Our study provides mechanistic perspectives regarding the role of NQO1 in CRC progression.