Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T.I., R.H., D.S., T.Y., H.O., Y.S., and Y.H. investigated the mechanisms involved in IL-26-mediated EGFR-TKI resistance in TNBC. We identified EphA3 as a novel functional receptor for IL-26 in TNBC. IL-26 induced dephosphorylation and downmodulation of EphA3 in TNBC, which resulted in increased phosphorylation of AKT and JNK against EGFR-TKI-induced endoplasmic reticulum (ER) stress, leading to tumor growth. Meanwhile, the blockade of IL-26 overcame EGFR-TKI resistance in TNBC. Since the gene encoding IL-26 is usually absent in mice, we utilized human transgenic (hIL-26Tg) mice as a tumor-bearing murine model to characterize the role of IL-26 in the differential effect of EGFR-TKI in human and mice and to confirm our in vitro findings. Our findings indicate that IL-26 activates the bypass pathway of EGFR-TKI, while blockade of IL-26 overcomes EGFR-TKI resistance in TNBC via enhancement of ER stress signaling. Our work provides novel insights into the mechanisms of EGFR-TKI resistance in TNBC via conversation of IL-26 with its newly identified receptor EphA3, while also suggesting IL-26 as a possible therapeutic target in TNBC. < 0.01 were considered significant and are indicated in the corresponding figures and physique legends. Supplementary information Revised Supplementary Figures(341M, pdf) Revised Legends to Supplementary Figures(119K, pdf) Acknowledgements AZ304 We thank members of Atopy (Allergy) Research Center (Juntendo University Graduate School of Medicine, Japan), members of the Laboratory of Morphology and Image Analysis, Research Support Center (Juntendo University Graduate School of Medicine, Japan), and members of the Laboratory of Cell Biology, Research Support Center (Juntendo University Graduate School of Medicine, Japan) for technical assistance and for the use of the experimental apparatus. Author contributions Conception and design: C.M. and K.O. Development of methodology: T.I, R.H., and H.O. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T.I., R.H., D.S., T.Y., H.O., Y.S., and Y.H. AZ304 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R.H., Y.H., Y.K., and K.O. Writing, review, and/or revision of the manuscript: T.I., R.H., N.H.D., C.M., and K.O. Review on English language as a native English speaker: N.H.D. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S.M., N.I., Y.H., and T.M.A. Study supervision: K.O. and AZ304 C.M. Funding This study was supported in part by a grant of the Ministry of Health, Labour, and Welfare, Japan (Grant Numbers 180101-01 (C.M.)), JSPS KAKENHI Grant Numbers JP19K21278 (T.I.), JP20K07683 (R.H.), JP20H03471 (C.M.), and JP18H02782 (K.O.). Data availability All data generated or analyzed during this study are included in this published article and its Supplementary HBEGF Information AZ304 files. Ethics approval AZ304 Animal experiments were conducted following protocols approved by the Animal Care and Use Committees at Juntendo University (300070). For clinical samples, human study protocols were approved by the Ethics Committees at Juntendo University Hospital (no: 17-252) and all specimens were collected after obtaining informed consent from the patients. All experiments were performed in accordance with relevant guidelines and regulations. Conflict of interest T.I., R.H, C.M., and K.O. are the patent holders of anti-IL26 mAbs. The remaining authors declare no competing interests. Footnotes Edited by J.-E. Ricci Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Takumi Itoh, Ryo Hatano Supplementary information The online version contains supplementary material available at 10.1038/s41419-021-03787-5..