Colon cancer is a therapy-resistant cancer with a low 5-year survival frequency. The drug 5-fluorouracil (5-FU) has been used as a first-line therapy in metastatic colon cancer in combination with leucovorin or oxaliplatin with a >40% resistance rate. High CysLTR expression in tumors is associated with poor survival of colon cancer patients. We sought to examine the role of CysLTR in 5-FU resistance and established 5-FU-resistant (5-FU-R) colon cancer cells. These 5-FU-R-cells expressed increased levels of CysLTR and showed increased survival and migration compared to nonresistant cells. Increases in thymidylate synthase and active β-catenin were also observed in the 5-FU-R-cells. LTD/CysLTR signaling was further increased and abolished after CYSLTR1 CRISPR-Cas9-knockdown and reduced in CysLTR-doxycycline-knockdown experiments and CysLTR-antagonist montelukast/5-FU-treated cells. Montelukast and 5-FU resulted in synergistic effects by reducing HT-29 cell and 5-FU-R-HT-29 cell migration and zebrafish xenograft metastasis. An increase in the stem cell markers in 5-FU-R-cells and 5-FU-R-cell-derived colonospheres and in CysLTR-Dox-knockdown cells increased colonosphere formation and stem cell markers was noticed after 5-FU treatment. IL-4-mediated stemness in both HT-29-colonospheres and 5-FU-R-cell derived colonospheres was abolished by montelukast or montelukast + 5-FU-treatment. Targeting CysLTR signaling by montelukast might reverse drug resistance and decrease resistance-derived stemness in colon cancer patients.