Proteus mirabilis is an important cause of urinary tract infections (UTIs) and can result in acute pyelonephritis. Proteus mirabilis expresses several, morphologically distinct, fimbrial species, and previous studies have shown that the nonagglutinating fimbriae (NAF) mediate bacterial adherence to a number of cell lines, including Madin-Darby canine kidney (MDCK) cells. Immunoblot overlay analysis of the plasma membrane fraction from MDCK cells with purified NAF revealed a 34-kDa band, which has been analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Database search identified galectin-3 as a potential protein candidate. Immunocytochemical assay of MDCK cells with a galectin-3-specific monoclonal antibody, anti-Mac-2, confirmed its presence on the plasma membrane extracellular surface. Preincubation of P. mirabilis with anti-Mac-2 monoclonal antibodies, specific for galectin-3, resulted in the inhibition of bacterial binding to MDCK cells. These data suggest a role for galectin-3, interacting with appropriately glycosylated surface receptors and P. mirabilis fimbriae, as a mediator of bacterial adherence in vitro.