Background and Aims: Scutellaria is one of the most popular traditional Chinese herbal remedies againstvarious human diseases, including cancer. In this study, we examined the active effects of Scutellaria extractand its main flavonoid constituents on the proportion of side population cells within human multiple myelomacell line RPMI8226 in vitro and explored the potential molecular mechanisms involved. Materials and Methods:The contents of flavonoids in ethanolic extract of Scutellaria baicalensis Georgi were determined using highperformance liquid chromatography. The antiproliferative effect of the ethanolic extract on RPMI-8226 wasdetermined by CCK assay. Apoptosis was measured by annexin combining with propidium iodide in a flowcytometer. Cell cycle analysis was performed by propidium iodide staining in combination with flow cytometryanalysis. Hoechst 33342 exclusion assay was used for the identification of side population within RPMI8226cells. The expression of ABCG2 protein was assessed by Western blotting assay. Results: The content of majorflavonoids constitutents of Scutellaria extract was baicalin (10.2%), wogonoside (2.50%), baicalein (2.29%), andwogonin (0.99%), respectively. The crude Scutellaria extract did not show significant anti-proliferative effect,apoptosis induction and cell cycle arrest in RPMI-8226 within the concentrations of 1-75μg/mL. However, theethanolic extract, baicalein, wogonin and baicalin reduced the side population cells in RPMI-8226, and datashowed that baicalein and wogonin had stronger inhibitory effects. Correspondingly, they also exhibited significanteffects on decreasing the expression level of ABCG2 protein in RPMI-8226 in vitro. Conclusions: Our results forthe first time demonstrated a novel mechanism of action for Scutellaria extract and its main active flavonoids,namely targeting SP cells by modulating the expression of ABCG2 protein. This study provides an insight fornew therapeutic strategies targeting cancer stem cells of multiple myeloma.