Epithelial cells lining the urinary tract are rich in globo series glycolipids, structurally defined by a Galalpha1,4Gal motif in the oligosaccharide moiety of this glycolipid family. This Galalpha1,4Gal motif is the attachment target for the P-fimbrial adhesin of uropathogenic Escherichia coli. We investigated the ability of a trisaccharide analog of this core motif, globotriose (Galalpha1,4Galbeta1,4Glc), to interfere with uropathogen attachment and colonization in vitro and in vivo. We assessed the ability of globotriose to inhibit and reverse the binding and agglutination of a P-fimbriated strain of E. coli (JR1) using human erythrocytes and immortalized human colonic epithelial cells as targets. Globotriose (5 mg/ml) completely inhibited and reversed cell agglutination and caused a 10- to 100-fold reduction in JR1 binding to target cells, as determined by flow cytometry. In preparation for an in vivo efficacy study, we investigated the distribution and pharmacokinetics of globotriose in the BALB/c mouse. Globotriose was administered via the tail vein, targeting an instantaneous plasma concentration of 5 mg/ml, and in a different experiment, animals were gavaged at 10 times the intravenous (i.v.) dose. Globotriose was rapidly cleared from plasma (half-life [t1/2], 6 min) and slowly excreted via the kidney (t1/2, 4 h). Urine levels of >5 mg/ml were maintained from 4 to 12 h after the i.v. bolus dose, which resulted in a 1-log reduction in established bladder colonization by JR1. These results suggest that free, soluble globotriose is a feasible alternative therapy for urinary tract infections.