Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.