Scutellaria barbata D. Don, a traditional Chinese medicine, reportedly possesses antitumor activity against a variety of tumors. In the present study, we investigated the cytotoxic effect of total flavonoids from S. barbata (TF-SB) on human hepatocarcinoma cells and the underlying molecular mechanisms regarding the effect were explored. TF-SB treatment significantly reduced the cell viability of human HCC MHCC97-H cells in a dose-dependent manner. Further flow cytometric analysis showed that the apoptosis rate of MHCC97-H cells increased and the mitochondrial membrane potential (∆ψm) of MHCC97-H cells decreased after TF-SB treatment. DNA ladder showed that TF-SB induced a significant increase in DNA fragmentation in MHCC97-H cells. Reverse transcription PCR and Western blot analysis revealed that the expression levels of Smac, Apaf-1, Cytochrome c, Caspase-9, and Caspase-3 were upregulated in a dose-dependent manner and after treatment with different concentrations of TF-SB for 48 h. These results suggest that TF-SB induces apoptosis in MHCC97-H cells through the mitochondrial pathway.