Background Baicalein, a natural flavonoid obtained from the Scutellaria baicalensis root, has been reported to inhibit growth of human lung cancer. However, the detailed mechanism underlying this has not been well elucidated. Methods Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Apoptosis was detected by flow cytometry analysis and caspase 3/7 assays. The expression of RUNX3 and FOXO3a mRNA were measured by real time RT-PCR methods. Western blot analysis was performed to measure the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPK alpha) and extracellular signal-regulated kinase 1/2 (ERK1/2), runt-related transcription factor 3 (RUNX3) and forkhead box O3a (FOXO3a). Silencing of FOXO3a and RUNX3 were performed by small interfering RNA (siRNA) methods. Exogenous expression of FOXO3a or RUNX3 was carried out by electroporated transfection assays. Results We showed that baicalein significantly inhibited growth and induced apoptosis of non-small cell lung cancer (NSCLC) cells in a time- and dose-dependent manner. Baicalein induced RUNX3 and FOXO3a protein expression, and increased phosphorylation of AMPKa and ERK1/2. Moreover, the inhibitors of AMPK and MEK/ERK1/2 reversed the effect of baicalein on RUNX3 and FOXO3a protein expression. Interestingly, while compound C had little effect on blockade of baicalein-induced phosphorylation of ERK1/2, PD98059 significantly abrogated baicalein-induced phosphorylation of AMPKa. Intriguingly, while silencing of RUNX3 abolished the effect of baicalein on expression of FOXO3a and apoptosis, silencing of FOXO3a significantly attenuated baicalein-reduced cell proliferation. On the contrary, overexpression of FOXO3a restored the effect of baicalein on cell growth inhibition in cells silencing of endogenous FOXO3a gene and enhanced the effect of baicalein on RUNX3 protein expression. Finally, exogenous expression of RUNX3 increased FOXO3a protein and strengthened baicalein-induced phosphorylation of ERK1/2. Conclusion Collectively, our results show that baicalein inhibits growth and induces apoptosis of NSCLC cells through AMPK alpha- and MEK/ERK1/2-mediated increase and interaction of FOXO3a and RUNX3 protein. The crosstalk between AMPKa and MEK/ERK1/2 signaling pathways, and the reciprocal interplay of FOXO3a and RUNX3 converge on the overall response of baicalein. This study reveals a novel mechanism for regulating FOXO3a and RUNX3 signaling axis in response to baicalein and suggests a new strategy for NSCLC associated targeted therapy.