The significance of DNA hydroxymethylation in stemness remains unknown. Here, we demonstrate 5hmC levels positively regulate mesenchymal stem cell (MSC) properties. Mechanistically, PARP1 recruits TET1 to hydrolyze methylated nucleotides on DNMT1 exons, helping CTCF to bind to exons and prevent DNMT1 alternative splicing in early MSCs. Furthermore, ATM phosphorylates TRIM37 at Th203 and promotes its entry into the nucleus, as well as the monoubiquitination of PARP1, thereby stabilizing the PARP1 protein. CTCF or TRIM37 knockdown induces replicative senescence of MSCs with loss of full-length DNMT1, while simultaneous treatment of MSCs during expansion with ATM activators, such as resveratrol, and TET1 activator, vitamin C, induces the rejuvenation of late MSCs through the TRIM37/PARP1/DNMT1 pathway. Through gene knockout, TRIM37 and PARP1 are shown to be involved in MSC aging and bone repair in vivo . This study highlights the role of DNA hydroxymethylation and its regulators in stemness, offering strategies for therapeutic interventions.