Objective This study investigates the regulatory role of pescadillo ribosomal biogenesis factor 1 (PES1) in cellular senescence and elucidates its underlying molecular mechanisms.Methods Using replicative senescence models of mouse embryonic fibroblasts (MEFs) and doxorubicin-induced senescence in human hepatocellular carcinoma HepG2 cells, we first quantified PES1 expression dynamics through immunoblotting. Subsequently, siRNA-mediated PES1 knockdown in HepG2 or other cells was employed to assess senescence phenotypes via β-galactosidase staining and immunodetection of senescence-associated markers. Mechanistic exploration involved Northern blot for pre-rRNA processing analysis and fluorescence microscopy for nucleolar morphology observation.Results PES1 expression was significantly downregulated in both replicatively senescent MEFs and doxorubicin-induced senescent HepG2 cells. siRNA-mediated PES1 depletion triggered premature senescence characterized by increased SA-β-gal positivity and upregulated p53/p21 signaling, while Rb pathway components remained unaltered. Notably, PES1 deficiency impaired 28S rRNA biogenesis and induced nucleolar fragmentation, indicative of nucleolar stress.Conclusion Inhibition of PES1 expression can induce nucleolar stress and activate p53-dependent rather than Rb-dependent senescence signals within cells.