Polysaccharides from genus Ganoderma have prominent anti-aging effect, but their mechanisms are incompletely clarifi ed. In our previous experiments, Ganoderma atrum polysaccharide (PSG) was exhibited to significantly alleviate senescence and DNA damage of A375 cells. To investigate its underlying mechanism, we conducted RNA-Seq analysis and found that PSG upregulated autophagy and mitochondria pathways compared to the model group. Further experiments showed that PSG relieved mitochondrial dysfunction via inhibiting the accumulation of reactive oxygen species (ROS) and loss of the mitochondrial membrane potential (MMP). Meanwhile, PSG activated autophagy and enhanced lysosomal function through transcription factor EB (TFEB). Interestingly, the pretreatment of autophagy inhibitors bafi lomycin A1 (Baf A1) reversed the ROS decline induced by PSG, which subsequently increased cellular senescence of A375. In addition, PSG had a similar anti-aging effect on normal fi broblast WI-38. Taken together, PSG might reduce ROS levels through activating autophagy, which in turn suppressed mitochondrial dysfunction and cellular senescence.