Nitidine chloride is a natural product. We synthesized novel nanoparticles of Nitidine chloride (TPGS-FA/NC), which evaluated anti-hepatocellular carcinoma potential capability in vitro and in vivo. Cell viability was assessed by MTT and colony assays; TPGS-FA/NC was examined by Confocal Microscopy though targeting Huh7 Hepatocellular Carcinoma Cells. A sphere culture technique was used to enrich Cancer Stem Cells (CSC) in Huh7 cells. The in vivo antitumor efficacy of TPGS-FA/NC was evaluated in Huh7 cell xenograft model, which were administered by TPGS-FA/NC for 2 weeks. TPGS-FA/NC (10, 20, 40 μg/mL) dose-dependently inhibited the proliferation of HCC cells. Interestingly, TPGS- FA/NC (10, 20, 40 μg/mL) drastically reduced the EpCAM+/CD133+ cell numbers, which suppressed the sphere formation and inhibited the expression of stem cell marker in the Huh7 spheroids. Additionally, TPGS-FA/NC time-dependently suppressed the AQP3/CD133/STAT3/JAK signalling pathways in Huh7 cells. In Huh7 cells xenograft bearing nude mice, TPGS-FA/NC administration significantly inhibited the tumour growth and markedly reduced the number of cancer stem-like cells in the tumours. TPGS-FA/NC treatment also reduced the expression of stem cell markers. The novel nitidine chloride nanoparticles markedly inhibited the HCC tumour growth through multiple mechanisms, and it may be a potential candidate drug for the therapy of hepatocellular carcinoma.