AbstractZinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) has long been known as a master transcriptional repressor of autophagy. Here, we identify a novel role for ZKSCAN3 in alleviating senescence that is independent of its autophagy-related activity. Downregulation of ZKSCAN3 is observed in aged human mesenchymal stem cells (hMSCs) and depletion of ZKSCAN3 accelerates senescence of these cells. Mechanistically, ZKSCAN3 maintains heterochromatin stability via interaction with heterochromatin-associated proteins and nuclear lamina proteins. Further study shows that ZKSCAN3 deficiency results in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, loss of heterochromatin, a more accessible chromatin status and consequently, aberrant transcription of repetitive sequences. Overexpression of ZKSCAN3 not only rescues premature senescence phenotypes in ZKSCAN3-deficient hMSCs but also rejuvenates physiologically and pathologically senescent hMSCs. Together, these data reveal for the first time that ZKSCAN3 functions as an epigenetic modulator to maintain heterochromatin organization and thereby attenuate cellular senescence. Our findings establish a new functional link among ZKSCAN3, epigenetic regulation, and stem cell aging.