Colon cancer has been proposed to be sustained by a small subpopulation of stem-like cells with unique properties allowing them to survive conventional therapies and drive tumor recurrence. Identification of targetable signaling pathways contributing to malignant stem-like cell maintenance may therefore translate into new therapeutic strategies to overcome drug resistance. Here we demonstrated that MEK5/ERK5 signaling activation is associated with stem-like malignant phenotypes. Conversely, using a panel of cell line-derived three-dimensional models, we showed that ERK5 inhibition markedly suppresses the molecular and functional features of colon cancer stem-like cells. Particularly, pharmacological inhibition of ERK5 using XMD8-92 reduced the rate of primary and secondary sphere formation, the expression of pluripotency transcription factors SOX2, NANOG, and OCT4, and the proportion of tumor cells with increased ALDH activity. Notably, this was further associated with increased sensitivity to 5-fluorouracil-based chemotherapy. Mechanistically, ERK5 inhibition resulted in decreased IL-8 expression and NF-κB transcriptional activity, suggesting a possible ERK5/NF-κB/IL-8 signaling axis regulating stem-like cell malignancy. Taken together, our results provide proof of principle that ERK5-targeted inhibition may be a promising therapeutic approach to eliminate drug-resistant cancer stem-like cells and improve colon cancer treatment.