Histone deacetylase (HDAC) inhibitors (HDIs) play an important role in the regulation of gene expression associated with cell cycle and apoptosis and have emerged as promising anticancer agents. In addition to their intrinsic anticancer properties, some studies have demonstrated that HDIs can modulate cellular responses to ionizing radiation (IR). Here we show evidence that co-treatment with the HDI trichostatin A (TSA) radiosensitizes human non-small cell lung cancer (NSCLC) A549 cells and H1650 cells. Cells were exposed to gamma-irradiation with or without TSA co-treatment. Clonogenic survival was significantly reduced in cells with TSA co-treatment. In A549 cells, TSA enhanced IR-induced accumulation of cells in G(2)/M phase, with upregulated expression of p21(waf1/cip1). In addition, TSA co-treatment caused pronounced apoptosis in irradiated cells, which was accompanied with p21(waf1/cip1) cleavage to a 15 kDa protein. The enhanced apoptotic effect was via mitochondrial pathway, as indicated by the increased dissipation of mitochondrial transmembrane potential (MMP) and release of cytochrome c from the mitochondria to the cytoplasm. Caspase-3 activation was also significantly increased, with accordingly more cleavage of PARP, associated with the repression of X-linked inhibitor of apoptosis protein (XIAP). Furthermore, TSA co-treatment impaired DNA repair capacity after IR by downregulation of Ku70, Ku80 and DNA-PKcs, reflected by enhanced and prolonged expression of gammaH2AX. Taken together, our results demonstrate that TSA acts as a powerful radiosensitizer in NSCLC cells by enhancing G(2)/M cell cycle arrest, promoting apoptosis through multiple pathways and interfering with DNA damage repair processes.