LysR-type transcriptional regulators (LTTRs) are ubiquitous and abundant among bacteria and control a variety of cellular processes. Here, we investigated the effect of Rsc1880 (a putative LTTR, hereafter designated PrhO) on pathogenicity of Ralstonia solanacearum. Deletion of prhO substantially reduced the expression of type III secretion system (T3SS) both in vitro and in planta, and resulted in significantly impaired virulence in tomato and tobacco plants. Complementary prhO completely restored the reduced virulence and T3SS expression to that of wild type. Moreover, the PrhO-dependent T3SS and virulence was conserved among R. solanacearum species. However, deletion of prhO did not alter biofilm formation, swimming mobility and in planta growth. Expression of some type III effectors was significantly reduced in prhO mutants, but the hypersensitive response was not affected in tobacco leaves. Consistent with the key regulatory role of HrpB on T3SS, PrhO positively regulated the T3SS through HrpB. Furthermore, PrhO regulated hrpB expression via two close paralogs HrpG and PrhG, which are two-component response regulators and positively regulate hrpB expression in a parallel way. However, deletion of prhO did not alter expression of phcA, prhJ and prhN, which are also involved in hrpB regulation. In addition, PrhO was expressed in a cell-density dependent manner, but negatively repressed its expression. No regulation was observed from HrpB, PhcA and PrhN to prhO expression. Taken all together, we genetically demonstrated that PrhO is a novel virulence regulator of R. solanacearum, which positively regulates the T3SS expression through HrpG and PrhG to HrpB and contributes to virulence. This article is protected by copyright. All rights reserved.