AbstractCancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N⁶,2’-O-dimethyladenosine (m⁶A_m) demethylase activity. While m⁶A_m is strategically located next to the m⁷G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m⁶A_m level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m⁶A_m methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m⁶A_m modification in stem-like properties acquisition. FTO-mediated regulation of m⁶A_m marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m⁶A_m modification and its potential adverse consequences for colorectal cancer management.