Nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) sometimes occurs in mildly fibrotic livers, while HCC incidence in NASH-related cirrhosis is lower than and less predictable than in hepatitis C virus (HCV)-related cirrhosis. Transforming growth factor (TGF)-β signaling in hepatocytic nuclei is implicated in fibrosis and carcinogenesis. TGF-βtype I receptor (TβRI) and c-Jun N-terminal kinase (JNK) differentially phosphorylate the mediator Smad3, resulting in 2 distinct phospho-isoforms: C-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). In mature hepatocytes, oncogenic signaling via the JNK/pSmad3L pathway antagonizes signaling via the tumor-suppressive TβRI/pSmad3C pathway. We immunohistochemically examined domain-specific Smad3 phosphorylation in liver biopsy specimens from 30 NASH patients representing different fibrotic stages and 20 chronically infected hepatitis C patients as controls, correlating Smad3 phosphorylation with clinical course. HCC occurred during follow-up in 11 of 12 NASH patients with abundant pSmad3L and limited pSmad3C but in only 2 of 18 with limited pSmad3L. In contrast, HCC developed in 12 of 15 NASH patients with limited pSmad3C but only 1 of 15 with abundant pSmad3C. Two of fourteen NASH patients with mild fibrosis developed HCC, their hepatocytic nuclei showed abundant pSmad3L and limited pSmad3C. Five of sixteen patients with severe fibrosis did not develop HCC, their hepatocytic nuclei showed limited pSmad3L and abundant pSmad3C. Smad phospho-isoforms may represent important biomarkers predicting HCC in NASH and potential therapeutic targets for preventing NASH-related HCC.