Galectin-3, a multifunctional lectin, is involved during cancer progression. Previous observations showed that both cytosolic expression and nuclear exclusion of galectin-3 in human prostate cancer cells were associated to progression of the disease. In this study, we examined the biological roles of galectin-3 when expressed either in the nucleus or in the cytosol. LNCaP, a galectin-3-negative human prostate cancer cell line, was used to generate transfectants expressing galectin-3 either in the nucleus or in the cytosol. No changes in cell morphology, proliferation, attachment to laminin-1 or androgen dependency were observed. Cytoplasmic galectin-3 induced significantly increased Matrigel invasion, anchorage-independent growth and in vivo tumor growth and angiogenesis, and decreased inducible apoptosis. Surprisingly, nuclear galectin-3 affected these parameters in an opposite fashion with an overall antitumoral activity. Thus, our study demonstrates that galectin-3 exerts opposite biological activities according to its cellular localization: nuclear galectin-3 plays antitumor functions and cytoplasmic galectin-3 promotes tumor progression.