Citrus canker, caused by the Gram-negative aerobic bacterium, Xanthomonas citri subsp. citri (Xac), is one of the most serious diseases for most commercial citrus cultivars and is responsible for significant economic losses worldwide. Secretion of type III effectors (T3Es) from the bacterial cytoplasm to host cell cytosol via a type III secretion system (T3SS) is crucial for virulence of most plant pathogenic bacteria. Studies on regulation of T3SS is of importance to broaden understanding of bacterial virulence and the plant-microbe interactions. In this study, we investigated the regulation of T3SS in X. citri. A conserved hypothetical protein, herein designated as VrpA (virulence-related periplasmic protein A), was identified as a critical factor for positive regulation of Xac T3SS. Based on several lines of experimental data, VrpA is required for efficient protein translocation via T3SS. Protein-protein interactions showed that VrpA physically interacts with periplasmic T3SS components: HrcJ and HrcC. However, mutation of VrpA did not affect T3SS gene expression. In addition, for the first time, we conducted a comparative analysis of Ser/Thr/Tyr Phosphoproteomes of Xac at in vitro and in planta. Eighty-nine phosphopeptides from 86 proteins were identified in both conditions, among which 46 phosphoproteins were identified in vitro, 33 phosphoproteins were found in planta and 7 phosphoproteins overlapped in both conditions. An ATP-dependent protease Lon was identified to be only phosphorylated in planta. Our genetic and biochemical experimental evidences demonstrated that Lon serves as a negative regulatory factor of T3SS in Xac. We found that Lon negatively regulates T3SS by targeting a key regulatory protein, HrpG. Phosphorylation attenuated Lon proteolytic activity, thus inhibited Lon-mediated HrpG degradation. We propose a phosphorylation switch model that controls Lon-mediated degradation of HrpG and regulates differential expression of type III secretion system in medium and plant. Our findings extend the understanding of T3SS regulation in plant pathogenic bacteria.