Obesity is a well-established risk factor for type 2 diabetes (T2D) and nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain incompletely understood. Here, we aimed to identify novel pathogenic factor and therapeutic target of liver metabolic dysfunctions.We applied tandem quantitative proteomics strategy to enrich and identify transcription factors (TFs) induced in the obese liver. We used flow cytometry of liver cells to analyze the source of the induced TF. We employed conditional knockout mice, shRNA, and small-molecule inhibitors to test metabolic consequences of the induction of identified TF. Finally, we validated mouse data in patient liver biopsies.We identified PU.1/SPI1, the master hematopoietic regulator, as one of the most up-regulated TFs in livers from diet-induced (DIO) and genetically obese (db/db) mice. Targeting PU.1 in the whole liver-but not hepatocytes alone-significantly improved glucose homeostasis and suppressed liver inflammation. Consistently, treatment with the PU.1 inhibitor DB1976 markedly reduced inflammation and improved glucose homeostasis and dyslipidemia in DIO mice, and strongly suppressed glucose intolerance, liver steatosis, inflammation, and fibrosis in a dietary NASH mouse model. Furthermore, hepatic PU.1 expression was positively correlated with insulin resistance and inflammation in liver biopsies from patients.These data suggest that the elevated hematopoietic factor PU.1 promotes liver metabolic dysfunctions, and may be a useful therapeutic target for obesity, insulin resistance/T2D, and NASH.