Objective: Epithelial-Mesenchymal Transition (EMT) plays an important role in the occurrence and development of pulmonary fibrosis and can lead to severe cell damage. Autophagy is a process that maintains cell balance by degrading and reusing damaged organelles, proteins, invading pathogens and other substances. To some extent, autophagy can protect cells, while uncontrolled and defective autophagy will further aggravate cell damage. Currently, it has been reported that autophagy can reduce the levels of apoptosis and mesenchymal transformation caused by certain pathogenic factors. Methods: Western blot and Real-time PCR were used to detect the changes in EMT, and immunofluorescence and Western blot were employed to detect the levels of autophagy. Then, autophagy was inhibited, and the protein and nucleic acid levels of EMT marker were detected. Finally, the changes in the markers of the Transforming Growth Factor-β/Smad pathway were detected after the addition of the autophagy inhibitor 3-Methyladenine (3-MA). Results: After stimulating A549 cells with pyocyanin for 24 hours, the protein and messenger RNA (mRNA) expression levels of the epithelial marker E-cadherin were significantly decreased compared with those of the control group, while the protein and mRNA expression levels of the mesenchymal marker α-Smooth Muscle Actin (α-SMA) were increased compared with those of the control group (P < 0.05). After inhibiting autophagy with 3-MA, the protein and nucleic acid expression levels of the autophagy marker LC3 and the epithelial marker E-cadherin were significantly decreased compared with those of the control group, while the protein and nucleic acid expression levels of the mesenchymal marker α-SMA were increased compared with those of the control group (P < 0.05). Studies have shown that the nucleic acid level of Transforming Growth Factor-β1 and the protein expression level of phosphorylated Smad2/3 in the group with the addition of the autophagy inhibitor 3-MA were significantly increased compared with those of the control group and the pyocyanin group (P < 0.05). Conclusion: Pyocyanin can induce EMT and autophagy in alveolar epithelial cells, and autophagy can inhibit the further development of EMT. It may inhibit the occurrence of EMT by reducing the activity of the Transforming Growth Factor-β/Smad pathway. These results indicate that inhibition of autophagy may enhance EMT by affecting the Transforming Growth Factor-β/Smad pathway.