The effect of fisetin on autophagy in hepatocellular carcinoma remains uncovered. HepG2 cells were exposed to different concentrations of fisetin (25, 50, and 100 µM) for 24 h. The cells were also treated with rapamycin and chloroquine alone or in combination with fisetin. Autophagic flux formation and ATP levels were determined. The changes in autophagic markers and AMPK signaling proteins were analyzed using qRT-PCR and Western blotting. Cyto-ID staining followed by flow cytometry showed that fisetin decreased autophagic flux formation in a dose-dependent manner. In gene expression analysis, the mRNA levels of mTOR, Atg5, Atg16L, and LC3A were elevated, whereas the mRNA levels of Atg7 and Beclin1 were downregulated in a dose-dependent manner compared to control. In the Western blotting analysis, fisetin treatment inhibited the expression of Atg7, Atg16L, mTOR, and pACC and elevated the expression of Atg5, AMPKα, AMPKβ1/2, ACC and Akt. Taken together, the results revealed that fisetin inhibited autophagy by the activation of PI3K/Akt/mTOR and modulation of AMPK signaling pathways. Our findings indicate that suppression of autophagy by fisetin may serve as an effective therapeutic strategy against HCC.