<p>Nonalcoholic steatohepatitis-associated hepatocellular carcinoma (NASH-HCC) is an increasing global health issue. Research indicates that amyloid precursor protein (APP) might be a diagnostic marker and therapeutic target for HCC. However, the specific role of O-glycosylation of APP in NASH-HCC remains unclear. A NASH-HCC mice model was established by feeding BALB/c inbred mice with a high-fat/high-cholesterol (HFHC) diet and injection of CCl₄. Hematoxylin–Eosin (HE) and Oil Red O (ORO) staining of liver tissue were then carried out to evaluate the progression of NASH-HCC. Immunohistochemistry (IHC) and Western blot were utilized to analyze APP levels. Immunoprecipitation (IP) was used to analyze the O-GalNAc modification of APP, and co-immunoprecipitation (Co-IP) and immunofluorescence were used to assess the Polypeptide N-Acetylgalactosaminyltransferase 18 (GALNT18) binding level of APP. Moreover, cell experiments confirmed the functional role of APP in HCC. APP was studied in vivo using an animal model. An up-regulation of APP and its O-glycosylation modification in patient-derived NASH-HCC tissues, animal models, and HCC cell lines was observed. The typical malignant phenotype of HCC, such as proliferation, migration, and invasion, was significantly suppressed by the knockdown of APP and was then partially rescued by the addition of UDP-GalNAc, a glycosylation activator. We demonstrated that GALNT18 interacted with APP in NASH-HCC models and upregulated its O-glycosylation. APP was finally shown to activate the EGR1/TGF-β1/Smad signaling, thereby driving the oncogenic progression of HCC. In vitro, APP knockdown inhibited NASH-HCC progression. This study identifies GALNT18-mediated O-glycosylation of APP as crucial in NASH-HCC development via the EGR1/TGF-β1/Smad pathway, suggesting that targeting GALNT18-APP signaling could be a therapeutic approach against NASH-HCC.</p>