: Cancer cells with 'stemness' are generally resistant to chemoradiotherapy. This study aims to compare the differences in radiation sensitivity of A549 and CD44A549 stem-like cells to X-rays and carbon ion radiation (C-ions), and to find a target that can kill cancer stem-like cells (CSCs) of non-small cell lung cancer (NSCLC). : The study used two cell lines (A549 and CD44A549). The tumorigenicity of cells was tested with animal experiments. The cells were irradiated with X-rays and C-ions. Cell viability was detected using the CCK-8 and EdU assay. A liquid chromatograph-mass spectrometer (LC-MS) helped detect metabolic differences. Protein and mRNA expression were detected using a Western blot, reverse transcription-quantitative (RT-qPCR), and PCR array. The autophagic activity was monitored with a CYTO-ID Autophagy Detection Kit 2.0. Immunofluorescence and co-immunoprecipitation helped to observe the localization and interaction relationships. First, we verified the radio-resistance of CD44A549 stem-like cells. LC-MS indicated the difference in autophagy between the two cells, followed by establishing a correlation between the radio-resistance and autophagy. Subsequently, the PCR array proved that TGM2 is significantly upregulated in CD44A549 stem-like cells. Moreover, the TGM2 knockdown by small interfering RNA could decrease the radio-resistance of CD44A549 cells. Bioinformatic analyses and experiments showed that TGM2 is correlated with the expression of CD44 and LC3B. Additionally, TGM2 could directly interact with LC3B. We established the CD44-TGM2-LC3 axis: CD44 mediates radio-resistance of CD44A549 stem-like cells through TGM2 regulation of autophagy. Our study may provide new biomarkers and strategies to alleviate the radio-resistance of CSCs in NSCLC.