Abstract Background/Aims: The global epidemic of obesity has led to an alarming rise in nonalcoholic steatohepatitis (NASH) and hepatocellular cancer (HCC). In addition to abnormal fat accumulation and liver injury, fibrosis is a key predictive factor for progression and transformation. Yet, our understanding of how a major fibrosis pathway, transforming growth factor β (TGF-β), and how members, SMAD3 with its adaptor SPTBN 1 -contribute to the progression of NASH-driven HCC remains unclear. Here, we sought to better understand the role of TGF-β1/SMAD3/SPTBN1 in regulating the switch between NASH and HCC. Methods: We generated liver-specific SPTBN1 knockout mice (Albumin Cre+Sptbn1loxp/loxp, LKO). LKO and controls were given a high-fat diet (HFD) or DEN; Phenotypic analyses and mechanistic insight were obtained through RNA-seq, mass spectrometry, structure modeling, cell fractionation, and imaging-immunofluorescence, immunohistochemistry, and interactions studies were performed in human HCC cell lines (Huh7, Hep3B). Results: Strikingly, LKO did not develop HFD induced obesity, NASH, and cancer, that occurred in wild type, Flox control, NASH, and DEN/HCC models. Compared to controls, LKO liver tissues displayed 2-7-fold decreases in NASH associated pro-fibrotic genes (Col1a1, Col1a2) and lipid metabolism genes (CD36, Slc27a1, Plin4, Plin2) (p;0.05), and over 4-fold increases in Fgf21 mRNA levels (LKO vs control: 3.67±1.9 vs 0.84±0.16). Analysis of key regulators of lipogenesis revealed decreased SREBP1 (0.33±0.11 VS 1.35±0.22, p=0.006) and targets as well as PPARG (3.15±1.24 VS 19.03±4.98, p=0.02) in LKO compared to controls. Functionally, Sptbn1 MEF cells show marked reduction in SREBP1 reporter activity compared to WT MEFs (LDLR-luc: 0.25±0.04 vs 1.0±0.08, p;0.05) with decreased nuclear SREBP1 labeling in LKO tissues. Depletion of Sptbn1 leads to impaired TGF-β1/SMAD3 signaling and decreased TNF-α induced ER stress/caspase-3 activated SREBP1. N-terminal SPTBN1 (D50-T975) binds SREBP1 (Q295-K374) and induces its nuclear translocation. HFD -driven caspase-3 activation and SREBP1-SPTBN1 cleavage and binding occur independently of insulin induced gene (INSIG) and SREBP cleavage-activating protein (SCAP). We observe raised levels of SPTBN1 together with Caspase-3, and lipogenic pathways (e.g., FASN, SCD1) in human NASH/HCC progression. Additional human HCC TCGA analysis revealed poor prognosis in patients with high SREBP1 activity. Finally, siRNA targeting Sptbn1 in vivo protects mice from HFD-induced NASH and reduces NASH-associated transcriptional changes in a human 3D-culture NASH model. Conclusions: We uncovered a surprising and promising role of SPTBN1 siRNA in inhibiting NASH and HCC, through modulation of its binding partners, SMAD3 and SREBP1. These data provide new insights into the switch in obesity driven liver cancer as well as new therapeutic targets. Citation Format: Shuyun Rao, Zhanhuai Wang, Kazufumi Ohshiro, Sobia Zaidi, Xiaochun Yang, Patricia Latham, Wilma Jogunoori, Xianyan Xiang, Inhee Chung, Kirti Shetty, Michele Vacca, Antonio Vidal-Puig, Lopa Mishra. A TGF-beta Pathway-SREBP1 axis controls liver diseases from nonalcoholic steatohepatitis to hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 89.