Galectin-3 is a member of a growing family of β-galactoside-binding animal lectins. Previous studies have demonstrated a variety of biological activities for this protein in vitro, including activation of cells, modulation of cell adhesion, induction of pre-mRNA splicing, and regulation of apoptosis. To assist in fully elucidating the physiological and pathological functions of this protein, we have generated galectin-3-deficient (gal3^−/−) mice by targeted interruption of the galectin-3 gene. Gal3^−/− mice consistently developed fewer inflammatory cell infiltrations in the peritoneal cavities than the wild-type (gal3^+/+) mice in response to thioglycollate broth treatment, mainly due to lower numbers of macrophages. Also, when compared to cells from gal3^+/+ mice, thioglycollate-elicited inflammatory cells from gal3^−/− mice exhibited significantly lower levels of NF-κB response. In addition, dramatically different cell-spreading phenotypes were observed in cultured macrophages from the two genotypes. Whereas macrophages from gal3^+/+ mice exhibited well spread out morphology, those from gal3^−/− mice were often spindle-shaped. Finally, we found that peritoneal macrophages from gal3^−/− mice were more prone to undergo apoptosis than those from gal3^+/+ mice when treated with apoptotic stimuli, suggesting that expression of galectin-3 in inflammatory cells may lead to longer cell survival, thus prolonging inflammation. These results strongly support galectin-3 as a positive regulator of inflammatory responses in the peritoneal cavity.