Few reports have been published on the potential role of autophagy in the efficacy of sonodynamic therapy (SDT). This study was to determine whether autophagy occurred after SDT and to investigate its relationship with apoptosis by performing inhibitor studies. In vitro murine sarcoma 180 (S180) cells were examined at different time points following SDT. Transmission electron microscopy (TEM) was used to identify the formation of autophagosomes. Western blots were used to assess the processing of LC3-I to LC3-II. Confocal microscopy was performed to reveal co-localization between mitochondria and autophagic vacuoles and re-distribution of apoptosis related proteins after sono-damage. Inhibitors of apoptosis and autophagy were used to determine the contributions of the two cellular responses to SDT efficacy. Autophagy was indentified by TEM observation of the presence of double-membrane delineated autophagic vesicles and by immunoblot observation of the increased LC3-II levels. The autophagy inhibitors, both 3-methyladenine (3-MA) and Bafilomycin A1 (Ba A1), were found to significantly enhance SDT-induced cell death. Blocking autophagy also led to increased dissipation of mitochondria potential, caspase-3 activity and the ultimate cell apoptosis. Whereas the pan-caspase inhibitor, z-VAD-fmk partially prevented SDT-induced cytotoxicity but did not obviously improve the autophagic vacuolization and mitochondria depolarization. This study suggests for the first time that autophagy participate in SDT-induced cell death and combination of SDT with autophagy inhibitors, especially preventing autophagy at the early stage by 3-MA, can significantly enhance the anti-tumor effect of SDT through induction of apoptosis and necrosis. Crown Copyright © 2010. Published by Elsevier Inc. All rights reserved.