Background: FL118, a novel camptothecin analogue, has been extensively studied in human cancers due to its superior antitumor properties. However, significantly less is known about its effect and underlying mechanisms in non-small cell lung cancer (NSCLC). Therefore, this study aimed to investigate the effects of FL118 on NSCLC cell growth, migration, invasion and apoptosis. Methods: CCK-8 assay was used to detect the effect of FL118 on the cell viability of A549 and H520 cells. Western Blot, Hoechst 33258 staining and Annexin V-FITC/PI double staining followed by flow cytometry were used to detect the effect of FL118 on apoptosis of A549 and H520 cells. Transwell assay and wound scratch experiment were used to detect the effect of FL118 on the invasion and migration of A549 and H520 cells. Immunofluorescence and Western Blot were applied to detect the effects of FL118 on the expression levels of EMT marked proteins E-cadherin and N-cadherin. Results: FL118 inhibited cell viability in a dose-dependent manner. Moreover, FL118 treatment led to a significant increase of apoptotic cells, cleaved PAPR, Bax, as well as the decrease of Bcl-2 level. FL118 treatment also dramatically reduced the migratory and invasive capabilities of NSCLC cells. Furthermore, FL118 reversed the epithelial to mesenchymal transition (EMT) process in A549 and H520 cells. Conclusion: FL118 could effectively suppress NSCLC cell growth, migration, invasion, and EMT process, while promoting apoptotic cell death.