8-Hydroxyquinaldic acid, the end-metabolite of tryptophan, is well-known metal chelator; however, its role in humans, especially in cancer promotion and progression, has not been fully revealed. Importantly, 8-hydroxyquinaldic acid is the analog of kynurenic acid with evidenced antiproliferative activity towards various cancer cells. In this study, we revealed that 8-hydroxyquinaldic acid inhibited not only proliferation and mitochondrial activity in colon cancer HT-29 and LS-180 cells, but it also decreased DNA synthesis up to 90.9% for HT-29 cells and 76.1% for LS-180 cells. 8-Hydroxyquinaldic acid induced changes in protein expression of cell cycle regulators (CDK4, CDK6, cyclin D1, cyclin E) and CDKs inhibitors (p21 Waf1/Cip1, p27 Kip1), but the effect was dependent on the tested cell line. Moreover, 8-hydroxyquinaldic acid inhibited migration of colon cancer HT-29 and LS-180 cells and increased the expression of β-catenin and E-cadherin. Importantly, antiproliferative and anti-migratory concentrations of 8-hydroxyquinaldic acid were non-toxic in vitro and in vivo. We reported for the first time antiproliferative and anti-migratory activity of 8-hydroxyquinaldic acid against colon cancer HT-29 and LS-180 cells.