Oxygen beams are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ- and oxygen ion-irradiation. Activation of various signaling molecules was looked in A549 lung adenocarcinoma cells irradiated with 2 Gy oxygen, 2 Gy or 6 Gy γ-radiation. Oxygen beam was found to be three times more cytotoxic than γ-radiation. By 4 h there was efficient repair of DNA in A549 cells exposed to 2 Gy or 6 Gy gamma radiation but not in cells exposed to 2 Gy oxygen beam as determined by γ-H2AX counting. Number of ATM foci was found to be significantly higher in cells exposed to 2 Gy oxygen beam. Percentage of cells showing ATR foci were more with gamma however number of foci per cell were more in case of oxygen beam. Oxygen beam irradiated cells showed phosphorylation of Chk1, Chk2 and p53. Many apoptotic nuclei were seen by DAPI staining in cells exposed to oxygen beam. The noteworthy finding of this study is the activation of the sensor proteins, ATM and ATR by oxygen irradiation and the significant activation of Chk1, Chk2 and p53 only in the oxygen beam irradiated cells.