Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to Nε-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.