The biological changes after irradiation in lung cancer cells are important to reduce recurrence and metastasis of lung cancer. To optimize radiotherapy of lung adenocarcinoma, our study systematically explored the mechanisms of biological behaviors in residual A549 and XWLC-05 cells after irradiation.Colony formation assay, cell proliferation assay, cell migration assay, flow cytometry, BALB/C-nu mice xenograft models and Western blot of pan-AKT, p-Akt380, p-Akt473, PCNA, DNA-PKCS, KU70, KU80, CD133, CD144, MMP2 and P53 were used in our study to assess biological changes after irradiation with 0, 4 and 8 Gy at 0-336 hr after irradiation in vitro and 20 Gy at transplantation group, irradiated transplantation group, residual tumor 0, 7, 14, 21, and 28 days groups in vivo.The ability of cell proliferation and radiosensitivity of residual XWLC-05 cells was better than A549 cells after radiation in vivo and in vitro. MMP-2 has statistical differences in vitro and in vivo and increased with the migratory ability of cells in vitro. PCNA and P53 have statistical differences in XWLC-05 and A549 cells and the changes of them are similar to the proliferation of residual cells within first 336 hr after irradiation in vitro. Pan-AKT increased after irradiation, and residual tumor 21-day group (1.5722) has statistic differences between transplantation group (0.9763, p=0.018) and irradiated transplantation group (0.8455, p=0.006) in vivo. Pan-AKT rose to highest when 21-day after residual tumor reach to 0.5 mm2. MMP2 has statistical differences between transplantation group (0.4619) and residual tumor 14-day group (0.8729, p=0.043). P53 has statistical differences between residual tumor 7-day group (0.6184) and residual tumor 28 days group (1.0394, p=0.007). DNA-PKCS has statistical differences between residual tumor 28 days group (1.1769) and transplantation group (0.2483, p=0.010), irradiated transplantation group (0.1983, p=0.002) and residual tumor 21 days group (0.2017, p=0.003), residual tumor 0 days group (0.5992) and irradiated transplantation group (0.1983, p=0.027) and residual tumor 21 days group (0.2017, p=0.002). KU80 and KU70 have no statistical differences at any time point.Different proteins regulated apoptosis, proliferation and metastasis of lung adenocarcinoma after radiotherapy at different times. MMP-2 might regulate metastasis ability of XWLC-05 and A549 cells in vitro and in vivo. PCNA and P53 may play important roles in proliferation of vitro XWLC-05 and A549 cells within first 336 hr after irradiation in vitro. After that, P53 may through PI3K/AKT pathway regulate cell proliferation after irradiation in vitro. DNA-PKCS may play a more important role in DNA damage repair than KU70 and KU80 after 336 hr in vitro because it rapidly rose than KU70 and KU80 after irradiation. Different cells have different time rhythm in apoptosis, proliferation and metastasis after radiotherapy. Time rhythm of cells after irradiation should be delivered and more attention should be paid to resist cancer cell proliferation and metastasis.© 2020 Dai et al.