Abstract Autophagy is frequently activated in cancer cells resistant to various apoptotic stimuli such as ionizing radiation, which supports the premise of enhancing the efficiency of radiotherapy by interventional means of autophagy regulation. In this study, we investigated whether autophagy pathway mediates the radio-sensitizing effect in head and neck cancer cells, and how the mechanism underlying the enhanced sensitivity works in cancer cells. The cellular responses following the ionizing radiation were examined by cell morphologic change, cell proliferation, clonogenic survival, cell cycle distribution, and expression level of several corresponding marker proteins in head and neck cancer HN-9 cells. Induction of autophagy was performed by down-regulating the mTOR signaling pathway using mTOR inhibitors such as rapamycin and PP242. Also, autophagy was inhibited by blocking the formation of autophagic vacuole (3-MA) or by disturbing the autophagosome maturation (bafilomycin A1, pepstatin A/E-46d). The change of autophagic flux was evidenced by the level of autophagy-related proteins and the morphological manifestation of autophagy. Upon exposure to radiation, the number of autophagic vacuoles was increased greatly in radiation dose- and time-dependent manners; however, the increase of apoptotic cells was not observed even in cells exposed to 12 Gy. In the meanwhile, the initial inhibition of cell cycle was maintained at slightly enhanced G1 phase. In some of the cases, it was restored to normal states, which implied that HN-9 cells adopted the mode of autophagy as a survival strategy against radiation. However, unexpectedly, further augmentation of autophagy pathway through the combination of either rapamycin or PP242 and radiation toward HN-9 cells hindered rather than enhanced clonogenic survival and cell proliferation, accompanying prolonged G1 arrest. Taken together, these results suggest that either induction of autophagy could serve as a therapeutic strategy to enhance radio-sensitivity, which is possibly due to irreversible cell cycle arrest following the excessive activation of autophagy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2011-3789