<p>5-Fluorouracil (5-FU) resistance in colorectal cancer (CRC) involves oxidative stress mechanisms, but the role of epitranscriptomic regulation remains unclear. This study investigates how oxidative post-translational modifications of the N6-methyladenosine (m⁶A) methyltransferase METTL16 contribute to 5-FU resistance. Parental (HCT8, HCT15) and 5-FU-resistant CRC cells were compared using redox proteomics, m⁶A-seq, RNA-seq, and functional assays. METTL16 S-glutathionylation was assessed via streptavidin pulldown and mass spectrometry. IGF2BP3’s role was validated through knockdown/overexpression, patient-derived organoids (PDOs), and xenograft models. Clinical relevance was evaluated in 112 CRC patient tissues and data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Drug synergy was tested using isoliquiritigenin (ISO) combined with 5-FU. Resistant cells exhibited elevated global protein S-glutathionylation and reduced m⁶A RNA methylation. METTL16 underwent site-specific S-glutathionylation at Cys548, leading to its degradation and subsequent m⁶A loss. Integrative omics identified IGF2BP3 as a key METTL16 target: reduced m⁶A on IGF2BP3 mRNA enhanced its nuclear export, splicing, and overexpression. IGF2BP3 knockdown sensitized resistant cells to 5-FU in vitro and in vivo, while overexpression conferred resistance. Mechanistically, IGF2BP3 destabilized CFTR mRNA (an ABC transporter), reducing 5-FU uptake. Clinically, high IGF2BP3 correlated with poor survival and 5-FU resistance in CRC patients. Targeting IGF2BP3 with ISO synergized with 5-FU, overcoming resistance in cells and xenografts. Oxidative stress-induced METTL16 S-glutathionylation drives 5-FU resistance by reducing m⁶A modification, enabling IGF2BP3 overexpression and CFTR suppression. IGF2BP3 is a biomarker of clinical resistance, and its targeting with ISO represents a promising combinatorial strategy to restore 5-FU efficacy in CRC.</p>