Constitutive activation of signal transducer and activator of transcription 3 (STAT3) is a common feature in human non-small cell lung cancer (NSCLC). STAT3 plays an important role in cancer progression as a driver oncogene and acquired resistance of targeted therapies as an alternatively activated pathway. W2014-S with pharmacophore structure of imidazopyridine, which was firstly reported to be utilized in STAT3 inhibitor discovery, was screened out as a potent STAT3 inhibitor from a library of small molecules. The aim of this study is to investigate the antitumor activities and mechanisms of W2014-S in NSCLC and effect on epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) resistance in vitro and in vivo. Methods: SPR analysis, Co-immunoprecipitation, confocal microscope imaging, and luciferase report gene assays were utilized to determine the mechanisms. Cell viability, colonial survival, wound healing, cell invasion assay, human cancer cell xenografts and PDX tumor xenografts were used to determine antitumor activities. Results: W2014-S disrupted STAT3 dimerization and selectively inhibited aberrant STAT3 signaling in NSCLC cell line. W2014-S strongly suppressed proliferation, survival, migration and invasion of lung cancer cells with aberrant STAT3 activation and inhibited the growth of human NSCLC cell xenografts and PDX tumor xenografts in mouse model. Furthermore, W2014-S significantly sensitized resistant NSCLC cell line to gefitinib and erlotinib in vitro and enhances the anti-tumor effect of gefitinib in TKI-resistant lung cancer xenografts in vivo. Conclusions: Our study has provided a novel STAT3 inhibitor with significant anti-tumor activities in NSCLC and suggests that combination of STAT3 inhibitor such as W2014-S with gefitinib could serve as a promising strategy to overcome EGFR-TKIs acquired resistance in NSCLC patients. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.