The aim of the present study was to elucidate the effects of ataxia telangiectasia mutated (ATM) kinase on the regulation of the extrinsic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2/DR5-mediated death pathway in human melanoma cells. We revealed that total ATM protein levels were high in some human melanoma lines compared with normal cells. The basal levels of active form ATM phospho-Ser¹⁹⁸¹ were also detectable in many melanoma lines and could be further up-regulated by γ-irradiation. Pretreatment of several melanoma lines just before γ-irradiation with the inhibitor of ATM kinase KU-55933 suppressed p53 and nuclear factor-κB (NF-κB) activation but notably increased radiation-induced DR5 surface expression, down-regulated cFLIP (caspase-8 inhibitor) levels, and substantially enhanced exogenous TRAIL-induced apoptosis. Furthermore, γ-irradiation in the presence of KU-55933 rendered TRAIL-resistant HHMSX melanoma cells susceptible to TRAIL-mediated apoptosis. In addition, suppression of ATM expression by the specific short hairpin RNA also resulted in down-regulation of cFLIP levels, up-regulation of surface DR5 expression, and TRAIL-mediated apoptosis in melanoma cells. Besides p53 and NF-κB, crucial regulators of DR5 expression, transcription factor STAT3 is known to negatively regulate DR5 expression. Suppression of Ser⁷²⁷ and Tyr⁷⁰⁵ phosphorylation of STAT3 by KU-55933 reduced STAT3 transacting activity accompanied by elevation in DR5 expression. Dominant-negative STAT3β also efficiently up-regulated the DR5 surface expression and down-regulated cFLIP levels in melanoma cells in culture and in vivo. Taken together, our data show the existence of an ATM-dependent STAT3-mediated antiapoptotic pathway, which on suppression sensitizes human melanoma cells to TRAIL-mediated apoptosis. [Cancer Res 2009;69(8):3510–9]