Aging is a complex process characterized by the gradual deterioration of cellular function and tissue integrity, leading to increased vulnerability to age-related diseases. Demethyleneberberine (DMB), a natural compound originally from Cortex Phellodendri Chinensis (CPC), is recognized for its anti-inflammatory properties and identified as a novel mitochondria-targeted antioxidant. This study aimed to explore the potential therapeutic efficacy of DMB in mitigating cellular senescence for antiaging research. We employed Nutlin-3a, a reversible inhibitor of the P53/MDM2 interaction, to induce cellular senescence in human embryonic lung fibroblast MRC-5 cells, followed by DMB treatment to evaluate its antagonistic effects. The findings indicated that DMB effectively counteracted Nutlin-3a induced cellular senescence by modulating the expression of age-related genes, enhancing mitochondrial function, reducing reactive oxygen species (ROS) levels, and influencing the cellular senescence-associated secretory phenotype (SASP). Mechanistically, DMB was able to directly activate FEN1, an intracellular DNA endonuclease involved in maintaining mitochondrial genomic stability and function. Inversely, knockdown of FEN1 markedly impairs the antiaging effects of DMB in senescent MRC-5 cells. Additionally, we further utilized C. elegans model to validate the antiaging effects of DMB in vivo. The results indicated that DMB obviously extend the lifespan of C. elegans under conditions of heat and oxidative stress by reducing ROS levels and lipid accumulation. In summary, our findings demonstrate the feasibility of exploiting a natural compound DMB to exert antiaging effects by activating FEN1 to improve mitochondrial function, thus providing novel therapeutic strategy for antiaging research and age-related diseases.