Non-small cell lung carcinoma (NSCLC) is the most common lung cancer with high morbidity and mortality. The traditional treatment for NSCLC is particularly liable to relapse with many side-effects. Barbaloin is a natural compound with anticancer efficacy. The present study aimed to investigate the anticancer potential of barbaloin in NSCLC. The results displayed that barbaloin inhibited the viability of A549 cells by decreasing cell growth and the expression level of Ki-67 and proliferating cell nuclear antigen (PCNA), especially at high concentrations (50 and 100 µM). Besides, barbaloin increased the apoptosis rate of A549 cells and induced an accumulation of G2/M phase. Increased expression of apoptosis-related proteins (caspase-3, -8 and -9) and the changed levels of cell cycle checkpoint proteins (p27, p53 and cyclin A) further convinced of the anti-viability effect of barbaloin in A549 cells. On the other hand, barbaloin significantly suppressed the invasion and migration of A549 cells, and restrained the expression of tumor metastasis-related proteins. We further explored the activation of pro-survival or pro-metastasis signaling pathways, including AKT, nuclear factor kappa B (NF-κB), mitogen-actived protein kinase (MAPK) and β-catenin. The results revealed that barbaloin inactivated the p38MAPK/Cdc25B/Hsp27 pathway by inhibiting p38 nucleus translocation, while no significant influence was observed among other pathways. Finally, barbaloin restrained the growth and hepatic metastases of A549 cells in vivo. Taken together, our research indicated that barbaloin inhibited the proliferation and metastasis of NSCLC cells in vivo and in vitro. This may provide safer and more effective aspects for the treatment of NSCLC.