The evpP gene in fish pathogen Edwardsiella tarda, coding the T6SS secretory protein EvpP and carrying an evpA-evpO independent promoter region, was crucial for host cell invasion. The transcription of evpP was positively regulated by either the two-component system EsrA-EsrB or iron concentration, and its overexpression was known to enhance the invasion ability in our previous study. This work demonstrated that the H-NS protein, a pleiotropic regulator of gene expression, was a new transcriptional modulator of evpP gene. The results showed that in vivo the transcriptional level of evpP was downregulated by H-NS and in vitro this global regulator interacted directly with evpP promoter region. Moreover, DNase I footprinting experiments mapping the interaction regions of H-NS and evpP revealed that this global regulator bound to evpP promoter and neighbouring areas at multiple sites. We provided a new insight into evpP regulation network and demonstrated the repression of H-NS to the transcription of evpP gene.