Galectin-3 (Gal3) has important roles in tumor transformation and metastasis. This study shows that c-Abl and Abl-related gene (Arg) associate with and phosphorylate Gal3. The SH (Src homology)3 domains of c-Abl/Arg bind to a P 80 GPPSGP motif of Gal3, and Tyr79 and Tyr118 are the major tyrosine phosphorylation sites. A consequence of this interaction and phosphorylation is the significant impairment of chaperone-mediated autophagy of Gal3. Cells expressing Gal3 and treated with the c-Abl/Arg inhibitor STI571, Gal3-depleted cells, and Gal3-depleted cells expressing Gal3 phosphorylation mutants all display an increased sensitivity to apoptosis-inducing agents. In addition, tumor cells expressing the phosphorylation mutants show impaired tumorigenicity. These results partially explain the antiapoptotic effect of Abl and Arg. As tumors frequently overexpress Gal3, a c-Abl/Arg-specific inhibitor may potentially be applied along with other antitumor drugs to target the lysosomal degradation of Gal3 in tumor therapy.