YbeY is a highly conserved RNase in bacteria and plays essential roles in the maturation of 16S rRNA, regulation of small RNAs (sRNAs) and bacterial responses to environmental stresses. Previously, we verified the role of YbeY in rRNA processing and ribosome maturation in Pseudomonas aeruginosa and demonstrated YbeY-mediated regulation of rpoS through a sRNA ReaL. In this study, we demonstrate that mutation of the ybeY gene results in upregulation of the type III secretion system (T3SS) genes as well as downregulation of the type VI secretion system (T6SS) genes and reduction of biofilm formation. By examining the expression of the known sRNAs in P. aeruginosa, we found that mutation of the ybeY gene leads to downregulation of the small RNAs RsmY/Z that control the T3SS, the T6SS and biofilm formation. Further studies revealed that the reduced levels of RsmY/Z are due to upregulation of retS Taken together, our results reveal the pleiotropic functions of YbeY and provide detailed mechanisms of YbeY-mediated regulation in P. aeruginosaIMPORTANCEPseudomonas aeruginosa causes a variety of acute and chronic infections in humans. The type III secretion system (T3SS) plays an important role in acute infection and the type VI secretion system (T6SS) and biofilm formation are associated with chronic infections. Understanding of the mechanisms that control the virulence determinants involved in acute and chronic infections will provide clues for the development of effective treatment strategies. Our results reveal a novel RNase mediated regulation on the T3SS, T6SS and biofilm formation in P. aeruginosa.