The nucleoid-associated protein H-NS can be either chromosomal- or plasmid-encoded. We provide in this report evidence indicating that chromosomal- and plasmid-encoded H-NS proteins may differ in their functional properties. The modulatory function of chromosomal H-NS is antagonized by the H-NSTEPEC protein. We show that the H-NS protein encoded by the IncHI plasmid R27 (H-NSR27) is less sensitive to H-NSTEPEC antagonism than its chromosomal counterpart. H-NSR27 plays a relevant role by modulating R27 conjugation in response to temperature. Hence, we suggest that this modulator has evolved to avoid the deregulation of R27 conjugation by H-NSTEPEC-like proteins.