Sepsis-induced lung injury (ALI) is a critical condition characterized by excessive immune responses and tissue damage. Previous evidence has underscored an upregulation pattern of DNA methyltransferase 1 (DNMT1) in sepsis. This study reveals the key role of DNMT1 in modulating regulatory T cell (Treg) activity in septic ALI. A septic mouse model was generated through cecal ligation and puncture. Treatment with either DNMT1 antagonist Thioguanine (ThG) or AAV-sh-DNMT1 significantly reduced immune cell infiltration, reduced production of pro-inflammatory cytokines, and increasing production of anti-inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) of mice, alongside improved lung pathology and integrity. Furthermore, the DNMT1 inhibition or silencing significantly enhanced population of FOXP3 Tregs in the BALF and lung tissue. Similar trends were observed in mice with specific DNMT1 deletion in CD4 T cells (DNMT1-CD4-ko). Regarding the mechanism, we observed that DNMT1 represses transcription of forkhead box O1 (FOXO1) by recruiting RUNX family transcription factor 1 (RUNX1) to the FOXO1 promoter. FOXO1-specific knockout in CD4 T cells reduced anti-inflammatory activity of Tregs. Additionally, administration of the CD25 antibody exacerbated sepsis-induced ALI in DNMT1-CD4-ko mice. Collectively, these findings illustrate that targeting DNMT1 interacts with RUNX1 to repress transcription of FOXO1, which reduces immunomodulatory activity of Tregs and augments inflammatory cascades in septic lung injury.