Horizontal gene transfer (HGT) plays a pivotal role in the evolution and adaptation of genome-reduced mycoplasmas. The conjugative properties of these organisms are key in this phenomenon but are largely understudied, particularly in vivo . In the present study, the ruminant pathogen Mycoplasma agalactiae was used as a model organism to document mycoplasma conjugation in environments of increasing complexity, from axenic to cell and organotypic culture conditions. Compared to axenic mating conditions, mycoplasma co-cultivation with goat epithelial cells or bovine precision-cut lung slices resulted in enhanced mating frequencies with high rates of M. agalactiae integrative and conjugative element (ICEA) self-dissemination. These results were conditioned by the presence of eukaryotic cells in the culture and influenced by competition between mating partners but were not limited to M. agalactiae , as similar results were observed with Mycoplasma bovis . Mycoplasma conjugation ex vivo was further characterized by analyzing mycoplasma chromosomal transfer (MCT), a newly discovered mechanism of horizontal exchange of chromosomal DNA that generates mosaic genomes. Compared to ICEA transfer, MCT was detected at lower rates under cell and organotypic culture conditions, suggesting a negative impact of these cellular environments on MCT or its progeny. Finally, mating experiments under nutrient-deprived conditions identified nucleotide stress as a potential factor influencing the modulation of mycoplasma conjugation by eukaryotic host cells. In conclusion, these results suggest that HGT in vivo is likely underestimated and provide valuable models to further study mycoplasma conjugation ex vivo . IMPORTANCE Conjugation is an evolutionary shortcut that bacteria use to exchange genetic information with their neighbors. Despite the fast evolution rate of the genome-reduced mycoplasmas, their conjugative properties remain largely understudied, particularly in vivo . Here we used the ruminant pathogen Mycoplasma agalactiae to study how mycoplasmas conjugate in co-culture with host-derived cells and tissues. Interestingly, conjugation was stimulated when mycoplasmas were co-cultured with eukaryotic cells. This was documented by monitoring the self-propagation of a mobile genetic element known as integrative and conjugative element (ICE) and the exchange of chromosomal DNA leading to the formation of mosaic genomes. While ICE transfer was observed at high frequency, only a few mosaic genomes were detected in the presence of eukaryotic cells. Further data point toward nucleotide stress as a possible factor modulating mycoplasma conjugation in cellular environments. These results suggest that mycoplasma-host interactions may stimulate conjugation in vivo .< Réduire