The acquisition of new traits through horizontal gene transfer depends on the ability of the recipient organism to express the incorporated genes. However, foreign DNA appears to be silenced by the histone-like nucleoid-structuring protein (H-NS) in several enteric pathogens, raising the question of how this silencing is overcome and the acquired genes are expressed at the right time and place. To address this question, we investigated transcription of the horizontally acquired ugtL and pagC genes from Salmonella enterica, which is dependent on the regulatory DNA-binding proteins PhoP and SlyA. We reconstituted transcription of the ugtL and pagC genes in vitro and determined occupancy of their respective promoters by PhoP, H-NS, and RNA polymerase in vivo. The SlyA protein counteracted H-NS-promoted repression in vitro but could not promote gene transcription by itself. PhoP-promoted transcription required SlyA when H-NS was present but not in its absence. In vivo, H-NS remained bound to the ugtL and pagC promoters under inducing conditions that promoted RNA polymerase recruitment and transcription of the ugtL and pagC genes. Our results indicate that relief of H-NS repression and recruitment of RNA polymerase are controlled by different regulatory proteins that act in concert to express horizontally acquired genes.