In a previous study, differential proteomic analysis was used to identify membrane proteins of the human enteropathogen , whose levels are influenced by OmpR, the transcriptional regulator in the two-component EnvZ/OmpR system. Interestingly, this analysis demonstrated that at 37 °C, OmpR negatively affects the level of over a dozen Ysc-Yop proteins, which constitute a type III secretion system (T3SS) that is essential for the pathogenicity of . Here, we focused our analysis on the role of OmpR in the expression and secretion of Yops (translocators and effectors). Western blotting with anti-Yops antiserum and specific anti-YopD, -YopE and -YopH antibodies, confirmed that the production of Yops is down-regulated by OmpR with the greatest negative effect on YopD. The RT-qPCR analysis demonstrated that, while OmpR had a negligible effect on the activity of regulatory genes and , it highly repressed the expression of . OmpR was found to bind to the promoter of the operon, suggesting a direct regulatory effect. In addition, we demonstrated that the negative regulatory influence of OmpR on the Ysc-Yop T3SS correlated with its positive role in the expression of , the master regulator of the flagellar-associated T3SS.