We aimed to evaluate the impact of a novel highly potent and selective serine-threonine DNA-dependent Protein Kinase inhibitor (M3814) on high-precision particle therapy of non-small cell lung cancer (NSCLC) with carbon ions (CI) and to study the effect of this combination therapy on tumor microenvironment (TME). The effectiveness of radiotherapy ± DNA-PK inhibitor (DI) was evaluated in NSCLC models in vitro and in vivo using clinical-like radiotherapy settings. Tumor growth was followed in human xenograft A549; murine syngeneic LLC and GEMM kRAS/p53 mutant s.c. NSCLC models after treatment with DI and X-rays. Additionally, irradiation with X-rays vs. CI ± DI was performed in the orthotopic LLC model and tumor response was evaluated by histopathology and transcriptome analysis. Standardized in vitro radiobiological pipeline including clonogenic survival, DNA damage response (DDR) analysis as well as functional invasion assays together with proteome profiling were performed. DDR inhibition by DI enhanced the effect of both low- and high- LET radiotherapy, i.e., X-rays and CI, respectively. Superior efficacy of CI vs. X-rays, and additional radiosensitizing effect by DI were observed in orthotopic LLC model. Histopathological analysis showed attenuation of tumor cell proliferation and epithelial–mesenchymal transition (EMT) after radiotherapy and DI, which was most apparent after CI treatment. The efficacy of combined treatment was not only reflected by impaired DDR inhibition, but also through attenuation of tumor invasion. DI affected invasion independently of DDR alteration, by decreasing AKT signaling. The relevance of DNA-PK (PRKDC) on tumor invasion was confirmed by CRISPR-Cas9 in A549 PRKDC knock out cells. Irradiation increased endostatin/COL18 production, which in turn suppressed the invasion and migration of A549 and H460 cells. While decrease of invasiveness by X-rays was dependent on the presence of endostatin, prevention of invasion by CI was less dependent on endostatin. The role of endostatin/COL18 in efficacy of radiotherapy was further confirmed in A549 COL18 knock down model. In addition to radiosensitizing and positive TME effects in the high dose region, synergistic anti-invasive activity of DI may facilitate eradication of disseminated tumor cells adjacent to gross tumor volume thereby reducing the uncertainties of underdosing in clinical target volume. Given the steep dose gradient of CI, these data provide novel rationales for widening the therapeutic window for high-precision high-LET irradiation.