Activation of autophagy is a hallmark in tumor cells treated with chemotherapy, but the role of autophagy in acquired resistance of lung adenocarcinoma to cisplatin-based chemotherapy remains to be clarified. Our aim was to address that question by surveying the autophagic activity in parental lung adenocarcinoma cell line A549 and its 8-fold, more resistant subcell line, A549/DDP, which was obtained by treating cisplatin with increasing concentrations. A549/DDP and A549 cells were exposed to serum-free culture medium or ionizing radiation. To measure the stress-induced autophagy, LC3-II, as an autophagosome marker, was measured by immunofluorescence and Western blotting. To determine the effect of 3-MA, a known inhibitor of autophagy, on overcoming acquired cisplatin resistance, the MTT assay and flow cytometry were performed. Western blotting analysis demonstrated that LC3-II was increased in A549/DDP cells, compared with those of parental A549 cells, under stress conditions. Meanwhile, immunofluorescence staining showed that LC3-II protein was located mainly in the cytoplasm of A549/DDP. We also found that 3-MA can enhance the growth inhibition and apoptotic effect of cisplatin in acquired resistant cells (A549/DDP). Collectively, our results provide evidence that the upregulation of autophagy plays a major role in cisplatin resistance of A549/DDP cells.