Radiotherapy plays a critical role in lung cancer treatment. Radiation can activate transforming growth factor-β (TGF-β) signaling and induce the epithelial-mesenchymal transition (EMT), which may lead to distant metastases. MicroRNAs (miRNAs) have been suggested to affect radiotherapy in lung cancer. miRNA Next-Generation Sequencing was performed to investigate the effects of irradiation on the miRNA profile of lung cancer A549 cells. The functions of identified miRNA on the radiation induced EMT and TGF-β activation in A549 cells were then explored. Protein expression was evaluated by western blotting. Immunofluorescence staining was performed to detect the localization of Snail. Luciferase Assay was used to determine the target gene regulated by the identified miRNA. Radiation time-dependently induced EMT in A549 lung cancer cells as indicated by the changes of morphology, the expression of EMT marker proteins (E-cadherin, α-SMA and Vimentin) and the nuclear localization of Snail. Moreover, miR-3591-5p was identified as the most significant increased miRNA in response to radiation, and further experiments indicated that miR-3591-5p was required for radiation induced EMT and TGF-β/Smad2/3 activation. Ubiquitin Specific Peptidase 33 (USP33) was a downstream target of miR-3591-5p as predicted by TargetScan and validated by 3’untranslated regions (UTRs) Luciferase Assay. USP33 could deubiquitinate PPM1A (protein phosphatase, Mg2+/Mn2+ dependent 1A), a phosphatase for Smad2/3. Ectopic expression of USP33 or PPM1A partially abolished the effects of miR-3591-5p on EMT and TGF-β/Smad2/3 activation. The present study revealed the critical role of miR-3591-5p/USP33/PPM1A in radiation-induced EMT via TGF-β signaling and may suggest novel radiation sensitise strategies for lung cancer.