Small regulatory RNAs (sRNAs) regulate multiple physiological functions in bacteria, and sRNA PrrH can regulate iron homeostasis and virulence. However, the function of PrrH in Pseudomonas aeruginosa (P. aeruginosa) bloodstream infection (BSI) is largely unknown. The aim of this study was to investigate the role of PrrH in P. aeruginosa BSI model. First, P. aeruginosa PAO1 was co-cultured with peripheral blood cells for 6 h. qRT-PCR results showed a transient up-regulation of PrrH expression at 1 h. Simultaneously, the expression of iron uptake genes fpvA, pvdS and phuR were upregulated. In addition, the use of iron chelator 2,2'-dipyridyl to create low-iron conditions caused up-regulation of PrrH expression, a result similar to the BSI model. Furthermore, the addition of FeCl3 was found to decrease PrrH expression. These results support the hypothesis that the expression of PrrH is regulated by iron in BSI model. Then, to clarify the effect of PrrH on major cells in the blood, we used PrrH mutant, overexpressing and wild-type strains to act separately on erythrocytes and neutrophils. On one hand, the hemolysis assay revealed that PrrH contributes to the hemolytic activity of PAO1, and its effect was dependent on the T3SS system master regulator gene exsA, yet had no association with the hemolytic phospholipase C (plcH), pldA, and lasB elastase genes. On the other hand, PrrH mutant enhanced the oxidative resistance of PAO1 in the neutrophils co-culture assay, H2O2-treated growth curve and conventional plate spotting assays. Furthermore, the katA was predicted to be a target gene of PrrH by bioinformatics software, and then verified by qRT-PCR and GFP reporter system. In summary, dynamic changes in the expression of prrH are iron-regulated during PAO1 bloodstream infection. In addition, PrrH promotes the hemolytic activity of P. aeruginosa in an exsA-dependent manner and negatively regulates katA to reduce the oxidative tolerance of P. aeruginosa.Copyright © 2023 Elsevier Ltd. All rights reserved.