Background. Self-transmissible Integrative and Conjugative Elements (ICE) are key in massive horizontal chromosomal exchanges observed in several mycoplasma species. Understanding the factors controlling ICE transfer from donor to recipient cells is thus of utmost importance to further control horizontal gene transfer (HGT) in these organisms. By using the ruminant pathogen M. agalactiae as a model system, we demonstrated that a major antigenic lipoprotein at the surface of the ICE-recipient partner is critical for ICE transfer from donor cells. Methods. Large-scale random transposon mutagenesis was combined with high-throughput mating assays to map in the chromosome of the ICE-recipient partner those regions that are facilitating HGT. Results. Up to 20 mutants were selected that identified 12 regions whose disruption was associated with a reduced mating frequency. Remarkably, 3 mutants all characterized by a transposon inserted at different positions of the P48 lipoprotein gene, were found fully resistant to ICE transfer from donor cells. The central role of this surface-exposed lipoprotein in ICE transfer was confirmed by complementation studies. Preliminary results suggest that the initial contact between mating partners might be mediated by the interaction between P48 and the ICE-encoded lipoprotein CDS14 expressed at the surface of the donor partner. Whether P48 may facilitate HGT in other species is unknown but this putative ABC transporter substrate-binding protein was found highly conserved across Mollicutes. Conclusion. These data suggest that not only ICE-donor cells but also recipient mating partners may modulate ICE dissemination and provide interesting opportunities to control ICE-mediated HGT in these organisms.