Objective To study if 5-fluorouracil (5-FU) can induce human lung adenocarcinoma A549 cells’ autophagy in order to provide theoretical basis for the tolerance of the chemotherapeutics. Methods Autophagic vacuole’s change was observed with acridine orange (AO) staining under fluorescent microscope and transmission election microscope(TEM); LC3-Ⅱ expression was tested with cellular immunochemistry; Apoptosis was detected by flow cytometry and AO staining with laser scanning confocal microscopy detection; Production of apoptosis’ laddershaped stripe was tested with DNA cataphoresis. Results After being treated with 5-fluorouracil, the cells cytoplasm appeared to have apparent autophagic vacuoles when observed with AO staining and TEM observations; The cells showed that LC3-Ⅱexpression was apparently increased and 3-MA significantly inhibited the increase of LC3-Ⅱ when observed with cellular immunochemistry; The cell apoptosis did not occur when tested by flow cytometry, AO staining and DNA ladder. Conclusion Low concentration（5μmol/L） of 5-fluorouracil can