Background and Aims: Metabolic dysfunction-associated steatotic liver disease (MASLD), driven by obesity and insulin resistance, is a growing cause of chronic liver disease which ultimately predisposes to hepatocellular carcinoma (HCC). We recently identified recurrent non-synonymous FOXO1-S22W mutations in MASLD livers. FOXO1 is a key insulin-regulated transcription factor that controls hepatic metabolism. Since S22W is known to resist insulin-induced nuclear export, we aimed to determine how it alters FOXO1 chromatin binding, interactome and transcriptional networks. Methods: We overexpressed GFP-tagged FOXO1-WT and FOXO1-S22W in PLC/PRF/5 cells and performed multi-omic analysis under insulin treatment: Anti-GFP ChIP-seq (FOXO1 binding), H3K4me1/H3K27ac ChIP-seq (enhancer activity), rapid immunoprecipitation mass spectrometry for endogenous proteins (RIME) (protein interactors of FOXO1 on chromatin), and RNA-seq (transcriptional output). Results: Both variants occupied ~30,000 chromatin sites basally. Insulin treatment caused WT-FOXO1 release, but FOXO1-S22W remained bound at 6,065 sites. While global H3K4me1 was stable, H3K27ac was dynamically altered, showing both gains and losses at S22W binding sites under insulin. Motif analysis confirmed FOXO1-S22W retained canonical forkhead motifs and selectively enriched co-binding with hepatocyte lineage factors (HNF4A/G, HNF1A) and AP-1/KLF motifs under insulin. RIME validated these changes, additionally showing S22W acquired binding to mitochondrial proteins while losing co-binding with E3 ubiquitin ligase under insulin, reversing the WT trend. RNA-seq showed genotype- and insulin-specific differential regulation of MASLD/HCC-linked genes (nuclear receptors, KLFs, JUN/FOS).