The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) induces apoptosis in a variety of human cancer cells including breast carcinoma and this property may be important for its chemopreventive and therapeutic effects. Resistance to 4HPR has been described, however, the molecular mechanisms underlying sensitivity or resistance to this retinoid are not clear. Recently, it has been shown that the carbohydrate-binding protein galectin-3, which has been implicated in tumor progression, contains the anti-death motif NWGR present in the anti-apoptotic protein Bcl-2. To determine whether galectin-3 expression can abrogate the effect of 4HPR, we tested the effects of 4HPR on apoptosis of cell clones derived from the galectin-3 deficient human BT549 breast carcinoma cells after transfection with either wild type galectin-3 (BT549Gal-3Wt), galectin-3 inactivated by a point mutation in the NWGR motif (BT549Gal-3Mu), or empty vector control (BT549Vec). Both BT549Vec and BT549Gal-3Mu cells showed a marked decrease in survival after treatment with 4HPR principally due to induction of apoptosis. 4HPR-induced apoptosis in these cells was associated with stimulation of reactive oxygen species generation, decreased levels of Bcl-2 protein, release of cytochrome c into the cytosol, increased caspase-3 activity, and poly(ADP-ribose) polymerase cleavage. In contrast, 4HPR failed to exert any of these effects in the BT549Gal-3Wt cells. The demonstration that galectin-3 suppresses 4HPR-induced apoptosis in human breast carcinoma cells suggests that the increased expression of galectin-3 during cancer progression may be associated with 4HPR resistance.