Tyrosine kinase inhibitors (TKI) against Bcr-Abl are the first-line therapeutics for chronic myelogenous leukemia (CML). However, the resistance to Bcr-Abl TKIs is induced in leukemic cells not only by loss of sensitivity to TKIs through Bcr-Abl-related molecular mechanisms but also by loss of addiction to Bcr-Abl TK activity by acquiring Bcr-Abl-unrelated additional oncogenic mutations. Therefore, the identification of an additional therapeutic target has been anticipated for achievement of a complete cure and to overcome resistance to treatment. We here showed that modified human Galectin-9 (hGal9), a lectin that show specific affinity for beta-galactosides, inhibits the proliferation of five CML-derived cell lines by inducing apoptosis at their IC(50)s from 17.5 to 164.9 nmol/L. Our study revealed that activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding protein family transcription factors, is the critical mediator for cell killing by hGal9, and that Noxa is one of the downstream effector molecules of ATF3. Bim, on the other hand, the BH3-only protein essential for apoptosis by Bcr-Abl TKIs, was not associated with hGal9-induced cell death. ATF3-mediated cell death by hGal9 was not hampered by the absence of p53, the presence of mutant Abl(T315I), or by P-glycoprotein overexpression. In addition, hGal9 showed the additive growth-inhibitory effect with imatinib on CML cell lines. Collectively, hGal9 is a candidate agent that may overcome various kinds of resistance to treatment for CML and may suggest that ATF3 may be a new target molecule for the development of new treatment modalities that can overcome resistance to currently available chemotherapeutics.