Hepatocellular carcinoma (HCC) is one of the common cancers in the world. Previous studies have shown that Notch1 contributes to growth, migration and invasion of HCC. Our previous studies have demonstrated that the transcription factor ZBP-89 can inhibit the proliferation and growth of HCC. However, the relationship between ZBP-89 and Notch1 is unknown. In this study, we aimed to study whether the inhibition of HCC by ZBP-89 was associated with Notch1 in HCC. The study was performed in HCC cells and tissues and a mouse tumor model was also employed to conform the results. We first measured the mRNA levels of representative genes involved in Notch, and related transcription factors in ZBP-89-overexpressing versus control cells. It was found that ZBP-89 overexpression significantly down-regulated the mRNA levels of Notch target genes including HES1, HEY1, HES6 and NRARP but not Hedgehog and Wnt molecules, suggesting that Notch signaling may be critical in ZBP-89-mediated inhibitory effects. This suggestion was supported by the negative correlation between ZBP-89 and HES1 in HCC tissues. We further observed that Notch1 but not Nocth2 and 3 was significantly down-regulated by ZBP-89 in tumor spheres and that Notch1 deficiency could lead to reduced tumor sphere forming and secondary colony forming capabilities of HCC cells. When Notch1 in ZBP-89-deficient HCC cells was blocked by its shRNA, the sphere formation induced by ZBP-89 depletion was prevented, demonstrating that Notch1 and ZBP-89 had opposite effects on liver cancer stem cells (LCSCs). Notch1 knockdown could also abrogate the secondary colony formation activities mediated by ZBP-89. NICD1 (Notch1 intracellular domain)- and MAML1-induced promoter activities of LCSC markers EpCAM and CD44 could be dose-dependently blocked by ZBP-89. Moreover, confocal microscope showed that ZBP-89 and NICD1 were co-localized in the nucleus and co-immunoprecipitation revealed that ZBP-89 formed a protein complex with NICD1 in the nucleus of HCC cells. Importantly, N-terminal (amino acids Δ6-180) of ZBP-89 was demonstrated to be essential for its binding with NICD1. Collectively, ZBP-89 inhibits LCSC self-renewal by binding to NICD1 in the nucleus to suppress the Notch1 pathway in HCC. This novel pathway identified should facilitate the development of new treatments to target LCSCs. (This study was supported by grants from the National Natural Science Foundation of China, No.81472339 and the Research Grants Council of HKSAR No. 14109516).