Sepsis is a severe syndrome caused by the imbalance of the host response to infection, accompanied by multiple organ damage, especially acute lung injury. SET Domain-Containing 2 (SETD2) is a methyltransferase catalyzing H3 lysine 36 trimethylation (H3K36me3) that regulates multiple biological processes. This study focused on explicating the action of SETD2 on macrophage function in sepsis and the precise mechanism involved. Enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting were used to determine expression. Luciferase reporter assay and chromatin immunoprecipitation assay were conducted to detect the binding of SETD2 or H3K36me3 with the hypoxia-inducible factor 1, alpha subunit (Hif1a) gene. A sepsis-induced acute lung injury model was constructed via cecal ligation and puncture (CLP). SETD2 was decreased in RAW 264.7 cells stimulated by lipopolysaccharide (LPS). Besides, SETD2 suppressed M1 macrophage polarization and glycolysis caused by LPS. HIF-1α was enhanced in RAW 264.7 cells stimulated by LPS and inversely related to SETD2 expression. In addition, SETD2-catalyzed H3K36me3 bound to the Hif1a gene to modulate HIF-1α expression. Furthermore, Hif1a silencing suppressed Setd2 silencing-induced M1 macrophage polarization and glycolysis in RAW 264.7 cells. Moreover, overexpression of Setd2 inhibited CLP-induced lung injury and M1 macrophage polarization in mice. SETD2 suppressed M1 macrophage polarization and glycolysis via regulating HIF-1α through catalyzing H3K36me3 in sepsis.