Background: Multiple myeloma (MM) currently remains largely incurable. Cancer stem cells (CSCs) are believed to be responsible for drug resistance and eventual relapse. In this study, we exploited a novel agent to evaluate its inhibitory effect on MM CSCs. Methods: Epirubicin (EPI)-loaded lipid microbubbles (MBs) conjugated with anti-ABCG2 monoclonal antibody (EPI-MBs + mAb) were developed and their effect on MM 138-CD34- CSCs isolated from human MM RPMI 8226 cell line plus ultrasound exposure in vitro and in vivo in a nonobese diabetic/severe combined immunodeficient mouse model were assessed. Results: EPI-MBs + mAb combined with ultrasound led to a significant decrease in the clone formation ability and the mitochondrial membrane potential along with an increase in MM CSC apoptosis. Moreover, treatment with EPI-MBs + mAb with ultrasound exposure remarkably inhibited the growth of MM CSC-derived tumors in xenograft nonobese diabetic/severe combined immunodeficient mice compared with a single agent or EPI-MBs + mAb without ultrasound exposure. The inhibitive efficacy was also correlated with an increased expression of caspase-3, Bax, and TUNEL and decreased expressions of PCNA, Bcl-2, and CD31. Conclusions: Our findings reveal that the EPI-MBs + mAb combined with therapeutic ultrasound may confer an effective approach for treatment of MM by induction of an apoptotic pathway in MM CSCs.Keywords: Cancer stem cells; Epirubicin; Microbubble; Multiple myeloma; Ultrasound.