Peritoneal fibrosis poses a significant challenge to the long-term efficacy of peritoneal dialysis (PD), with emerging evidence highlighting the role of cellular senescence in its pathogenesis. p16 is a cell cycle regulator that has been implicated in cellular senescence. Mixed-lineage leukemia 1 (MLL1) forms a complex with WD-40 repeat protein 5 (WDR5) and exhibits histone H3K4 methyltransferase activity. We have previously shown that inhibition of the MLL1/WDR5 complex reduces p16 expression and attenuates renal senescence after injury in mice. This study aimed to investigate whether inhibiting MLL1/WDR5 attenuates peritoneal senescence, inflammation, and fibrosis in both human samples and in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO-injected mice), while also exploring the associated underlying mechanisms. MLL1/WDR5, histone 3 lysine 4 trimethylation (H3K4me3), and p16 expression were elevated in TGF-β1-stimulated human peritoneal mesothelial cells (HPMCs), non-adherent cells obtained from patients undergoing PD, and the submesothelial compact zones of MGO-injected mice. Notably, p16 expression in these cells was positively correlated with the dialysate/plasma creatinine ratio. Treatment with the MLL1/WDR5 protein-protein interaction inhibitors MM-102 and OICR-9429 reduced H3K4me3 levels and p16 expression, suppressing fibrosis in HPMCs as well as peritoneal fibrosis and inflammation in MGO-injected mice. These inhibitors also improved peritoneal function in MGO-injected mice. Additionally, we demonstrated that MLL1/WDR5-induced H3K4me3 directly regulates p16 gene transcription, and that inhibiting MLL1/WDR5 reduces H3K4me3, thereby suppressing p16 gene transcription. These findings suggest that targeting MLL1/WDR5 activation alleviates peritoneal senescence, inflammation, and fibrosis, highlighting its potential as a promising therapeutic strategy for peritoneal fibrosis.