We previously identified EMICORON as a novel G-quadruplex (G4) ligand showing high selectivity for G4 structures over the duplex DNA, causing telomere damage and inhibition of cell proliferation in transformed and tumor cells. Here, we evaluated the antitumoral effect of EMICORON on advanced models of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies, demonstrated that EMICORON had an unprecedented antitumor activity warranting further studies of EMICORON-based combination treatments.