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Peptidoglycan hydrolases are enzymes responsible for breaking the peptidoglycan present in the bacterial cell wall, facilitating cell growth, cell division and peptidoglycan turnover. <i>Xanthomonas citri</i> subsp. <i>citri</i> (<i>X. citri</i>), the causal agent of citrus canker, encodes an <i>Escherichia coli</i> M23 peptidase EnvC homolog. EnvC is a LytM factor essential for cleaving the septal peptidoglycan, thereby facilitating the separation of daughter cells. In this study, the investigation focused on EnvC contribution to the virulence and cell separation of <i>X. citri</i>. It was observed that disruption of the <i>X. citri envC</i> gene (Δ<i>envC</i>) led to a reduction in virulence. Upon inoculation into leaves of Rangpur lime (<i>Citrus limonia</i> Osbeck), the <i>X. citri</i> Δ<i>envC</i> exhibited a delayed onset of citrus canker symptoms compared with the wild-type <i>X. citri</i>. Mutant complementation restored the wild-type phenotype. Sub-cellular localization confirmed that <i>X. citri</i> EnvC is a periplasmic protein. Moreover, the <i>X. citri</i> Δ<i>envC</i> mutant exhibited elongated cells, indicating a defect in cell division. These findings support the role of EnvC in the regulation of cell wall organization, cell division, and they clarify the role of this peptidase in <i>X. citri</i> virulence.