Beauvericin (BEA) is a cyclic hexadepsipeptide that derives from Codyceps cicadae. Our previous study results indicated that the cytotoxic effects of BEA on human A549 lung cancer cells BEA occur through an apoptotic pathway, which involves the up-regulation of cytochrome c release from mitochondria, upregulation of caspase 3 activity, and cellular and morphological changes. In this study, we identified that the mitogen-activated protein kinase (MAPK) inhibitor U0126 inhibits the cytotoxic effects of BEA on A549 cells. After exposing human A549 cells to 10 μM BEA, we observed a significant and dose-dependent increase in the percentage of hypoploid (sub-G1) phase cells in the A549 population. Following the pretreatment of the A549 cells with 25 μM U0126, the distribution of A549 cells in the sub-G1 phase decreased significantly. The BEA treatment resulted in a significant increase apoptosis in A549 cells by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, the MEK1/2 (mitogen-activated protein kinase kinase)-ERK42/44 (extracellular signal-regulated kinases)-90RSK (ribosomal s6 kinase) signaling pathway was activated in BEA-induced apoptotic A549 cells. Furthermore, treatment with MEK1/2 inhibitor U0126 was capable to attenuate the BEA induced typical apoptotic morphological change, apoptotic cells, and MEK1/2-ERK42/44-90RSK signaling pathway. These results suggested that MEK1/2-ERK42/44-90RSK signaling pathway may play a important role in BEA-induced apoptosis in human NSCLC A549 cancer cells.