The phytopathogen Agrobacterium tumefaciens C58 expresses two lactonases, AttM and AiiB. We showed that expression of the aiiB gene was controlled by agrocinopines A and B and required the agrocinopine-ABC transporter Acc, but was not affected by the level of quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8-HSL). In the presence of agrocinopines, a constructed aiiB mutant accumulated OC8-HSL at a level 10-fold higher than that of the wild-type strain, and showed an exacerbated expression of a key QS-regulated function, conjugation of Ti plasmid (in vitro and in planta), as well as an increase of the number of emerging tumors on the host plant. The expression and acyl-HSL-degrading activity of AttM were evident in the presence of wounded tissues; however, in unwounded plant tumors, the QS-regulated functions were weakly affected in an attM mutant. By contrast, we observed that attM conferred a selective advantage in the course of colonization of plant tumors. Finally, polymerase chain reaction survey of genes attM and aiiB showed that they were not strictly conserved in the genus Agrobacterium. This work proved that the lactonases AttM and AiiB are regulated by different plant signals and are implicated in different functions in the course of the A. tumefaciens C58-host interaction.