Sepsis-induced acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) remains the leading complication for mortality caused by bacterial infection. The regulatory T (Treg) cells appear to be an important modulator in resolving lung injury. Despite the extensive studies, little is known about the role of macrophage HMGB1/PTEN/β-catenin signaling in Treg development during ALI. Objectives: This study was designed to determine the roles and molecular mechanisms of HMGB1/PTEN/β-catenin signaling in mediating CD4⁺CD25⁺Foxp3⁺ Treg development in sepsis-induced lung injury in mice. Setting: University laboratory research of First Affiliated Hospital of Anhui Medical University. Subjects: PTEN/β-catenin Loxp and myeloid-specific knockout mice. Interventions: Groups of PTEN^loxp/β-catenin^loxp and myeloid-specific PTEN/β-catenin knockout (PTEN^M-KO/β-catenin^M-KO) mice were treated with LPS or recombinant HMGB1 (rHMGB1) to induce ALI. The effects of HMGB1-PTEN axis were further analyzed by in vitro co-cultures. Measures and Main Results: In a mouse model of ALI, blocking HMGB1 or myeloid-specific PTEN knockout (PTEN^M-KO) increased animal survival/body weight, reduced lung damage, increased TGF-β production, inhibited the expression of RORγt and IL-17, while promoting β-catenin signaling and increasing CD4⁺CD25⁺Foxp3⁺ Tregs in LPS- or rHMGB-induced lung injury. Notably, myeloid-specific β-catenin ablation (β-catenin^M-KO) resulted in reduced animal survival and increased lung injury, accompanied by reduced CD4⁺CD25⁺Foxp3⁺ Tregs in rHMGB-induced ALI. Furthermore, disruption of macrophage HMGB1/PTEN or activation of β-catenin significantly increased CD4⁺CD25⁺Foxp3⁺ Tregs in vitro. Conclusions: HMGB1/PTEN/β-catenin signaling is a novel pathway that regulates Treg development and provides a potential therapeutic target in sepsis-induced lung injury.