Sensitization of mismatch repair (MMR)-deficient colorectal cancer (CRC) cells by 5-Fluorouracil (5-FU) is well-documented. But not much is known about the treatment of MMR-proficient CRC cancer stem cells (CRC-CSCs). Here, we investigated whether a PARP inhibitor (ABT-888) can enhance the 5-FU-mediated apoptosis in CRC-CSCs through MMR pathway inhibition. The anti-cancer action of 5-FU+ABT-888 combination in CRC-CSCs has been studied by using in vitro, ex vivo, and in vivo preclinical model systems. 5-FU caused DNA damage in CRC-CSCs, and ABT-888 enhanced the accumulation of DNA mismatches by downregulating the MMR pathway, triggering S-phase arrest, and finally apoptosis and cell death in 5-FU-pre-treated MMR-proficient-CRC-CSCs at much lower concentrations than their individual treatments. After 5-FU treatment, PARylated-PARP1 activated MMR pathway by interacting with MSH6. But, upon ABT-888 treatment in 5-FU-pre-exposed CSCs, PARylation was inhibited, as a result of which PARP1 could not interact with MSH6, and other MMR proteins were downregulated. The role of MSH6 in PARP1-mediated MMR activation, was confirmed by silencing MSH6 gene, which resulted in MMR pathway shutdown. Similar results were obtained in ex vivo and in vivo model systems. 5-FU+ABT-888 combination enhanced CRC-CSCs death by increasing DNA damage accumulation and simultaneously inhibiting the MMR pathway in MMR-proficient cells. But this study does not discuss whether the combination treatment will increase the sensitivity of MMR-deficient CSCs, for which further research will be performed in the future.