Fractionated ionizing radiation (FIR) is a radiotherapy regimen that is regularly performed as part of lung cancer treatment. In contrast to the growth inhibition caused by DNA damage, immunomodulation in post-irradiated cancer cells is not well documented. Interferon (IFN)-γ confers anticancer activity by triggering both growth inhibition and cytotoxicity. This study investigated the priming effects of FIR with immunomodulation on the anticancer IFN-γ. Cell morphology, cell growth, and cytotoxicity were observed in FIR-treated A549 lung adenocarcinoma. Induction of p53 and epithelial-mesenchymal transition (EMT) were monitored. Following FIR, activation of IFN-γ signaling pathways were detected. FIR caused changes in cell morphology, inhibited cell growth, and induced cytotoxicity. While p53 was induced by FIR, no epithelial-mesenchymal transition could be found. Following IFN-γ stimulation, FIR-induced p53-associated cell cytotoxicity was significantly enhanced. Additionally, FIR increased the downstream response to IFN-γ by facilitating IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) signaling without affecting the receptor expression. FIR-facilitated STAT1 activation through the mechanism involving mitogen-activated protein kinase activation and Src-homology 2 domain-containing tyrosine phosphatase 2 inactivation. These results demonstrate the FIR-facilitated IFN-γ signaling and its anticancer activity.