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Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter P exsA and its operon promoter P exsC . Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the P exsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the P exsC promoter to repress the expression of T3SS genes. The repression of MvaT on P exsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Yin, Liu, Pan, Lv, Bai, Bai, Cheng, Wu, Ha and Jin.)