Lung cancer represents the leading cause of cancer-associated mortality, and non-small-cell lung cancer (NSCLC) is the most frequent histologic sub-type. It is therefore urgent to develop novel agents for the treatment of NSCLC. Fraxetin (FXT) is a potent plant-derived product and has been recognized as a promising anticancer agent for breast cancer and osteosarcoma. However, the anti-cancer potential of FXT for NSCLC remains to be elucidated. Accordingly, in the present study, we evaluated the inhibitory effect of FXT on the proliferation and growth of NSCLC cells using six human NSCLC cell lines: A549, H460, HCC827, H1650, PC-9 and H1975. FXT exhibited significant inhibitory effects on the proliferation of these cancer cell lines. By contrast, no inhibitory effect was observed on the viability of non-cancer lung cell lines even at the highest concentration of FXT (100 mu M). Among the NSCLC cell lines, HCC827 and H1650 cells showed the most sensitive to FXT. Accordingly, HCC827 and H1650 cells were used for the subsequent experiments. Flow cytometric analysis revealed that FXT caused a significant cell cycle arrest and pro-apoptotic effects. Mechanistically, FXT suppressed the IL-6-induced phosphorylation of tyrosine residue (Tyr705) of signal transducer and activator of transcription 3 (STAT3) probably by binding to STAT3. Molecular docking and molecular dynamic simulations studies indicated that FXT interacts with STAT3 through hydrogen bond and hydrophobic interaction. In conclusion, these findings suggest that FXT could be a promising lead compound to be used as a novel STAT3 inhibitor and potential antitumor agent for the treatment of NSCLC.