Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to identify the association between miR-138 and Sentrin/SUMO-specific protease 1 (SENP1) as a radiosensitization-related gene and characterize the biological function by which SENP1 was regulated by miR-138 to influence radiosensitization in lung cancer cells. In this study, we showed that miRNA-138 is reduced in both lung cancer clinical specimens and cell lines and is effective to inhibit SENP1 expression. Moreover, high levels of miR-138 are associated with lower levels of lung cancer cell proliferation and colony formation. Then, we investigated the underlying mechanisms responsible for the increase in the radiosensitivity of lung cancer cells when SENP1 is inhibited by miR-138. We further show that the increased radiosensitivity may be the result of an increased γ-H2AX expression, an increased rate of apoptosis, and changes in the cell cycle. In conclusion, our data demonstrate that the miR-138/SENP1 cascade is relative to radiosensitization in lung cancer cells and is a potential radiotherapy target.