Mobile genetic elements drive bacterial evolution by promoting genetic plasticity and adaptation; among them, mycoplasma Integrative and Conjugative Elements (ICEs) challenge the notion that mycoplasmas evolved solely through genome reduction. However, they remain poorly characterized, particularly in Mycoplasma hominis, which is a human genital pathogen. In this study, we investigated the circularization, transcription, and protein expression of M. hominis 4788 ICE (ICEHo4788) under the following four environmental conditions: axenic growth, coculture with HeLa cells, mitomycin C exposure, and thermal stress. During axenic growth, ICEHo4788 circularization peaked at 12 h with a 9.4-fold increase in the number of circular forms. Mitomycin C and cold shock induced only a moderate increase in circularization (approximately 3-fold), whereas heat shock significantly reduced the number of circular forms. In coculture with HeLa cells, a strong increase in the number of circular forms was observed at 72 h and 7 days postinfection with 10- and 23-fold increases, respectively. Coculture also led to a 5- to 23-fold increase in transcription at 7 days, whereas axenic growth, mitomycin C exposure, and thermal stress did not differ. Proteomic analysis revealed that MhoH(a), CDS18, CDS17, and CDS11 were significantly overexpressed at 12 h. Neither mitomycin C nor cold shock induced changes in ICE-related proteins, but heat shock upregulated two M. hominis molecular chaperones and ICEHo4788 MhoH(a). In conclusion, environmental conditions modulate M. hominis ICE activity with strong stimulation in the presence of eukaryotic cells. These findings offer new insights into the regulation and environmental responses of M. hominis ICEs.Read less <