The intestine expresses metabolic enzymes and transporters and functions as a barrier to orally administered xenobiotics. This study aimed to examine the importance of multidrug resistance-associated protein 3 (Mrp3/Abcc3) in the serosal efflux of glucuronide conjugates formed in the intestine using wild-type and Mrp3(-/-) mice. The everted sacs of the intestine were incubated with 4-methylumbelliferone (4MU), and the efflux rates of intracellularly formed glucuronide conjugate of 4MU (4MUG) into the mucosal and serosal sides were determined. The permeability-surface area product across the serosal membrane (PS(serosal)) of 4MUG in wild-type mice was greatest in the duodenum followed by the jejunum, ileum, and colon. The corresponding parameters were significantly reduced in Mrp3(-/-) mice (approximately 33% of that in wild-type mice) except for the colon where the PS(serosal) of 4MUG was similar between wild-type and Mrp3(-/-) mice. There was no difference in the PS(mucosal) of 4MUG in whole segments of the intestine between wild-type and Mrp3(-/-) mice. In addition to 4MUG, the PS(serosal) of the glucuronide conjugates of 7-ethyl-10-hydroxycamptothecin (SN-38) and acetaminophen in the jejunal everted sacs were also significantly reduced in Mrp3(-/-) mice compared with wild-type mice. There was no difference in the mRNA and protein expression of efflux transporters between wild-type and Mrp3(-/-) mice. These results suggest that Mrp3 plays major roles in the efflux transport of various glucuronide conjugates from the enterocytes to the portal blood in the small intestine together with unknown transporter(s), but the contribution of Mrp3 to the basolateral efflux of 4MUG was negligible in the colon.