ABSTRACT Xanthomonas campestris pv. campestris ( Xcc ) and X. oryzae pv. oryzae ( Xoo ) are crucial plant pathogenic bacteria, causing crucifer black rot and rice leaf blight, respectively. Both bacterial species encode a protein containing the YiiD_C domain, designated MadB, which exhibits an 87.5% sequence identity between their MadBs. The madB genes from either Xoo or Xcc successfully restored the growth defect in Ralstonia solanacearum and Escherichia coli fabH mutants in vivo. In vitro assays demonstrated that MadB proteins possess malonyl-ACP decarboxylase activity, although Xcc MadB exhibited lower activity compared with Xoo MadB. Mutation of madB in both Xoo and Xcc strains led to decreased pathogenicity in their respective host plants. Interestingly, the Xoo madB mutant exhibited a significant increase in branched-chain fatty acid production, whereas the Xcc madB mutant showed only minor changes in fatty acid composition. Despite the reduction in exopolysaccharide (EPS) synthesis due to madB mutation in both Xoo and Xcc , EPS production in the Xoo madB mutant could be restored by exogenous sodium acetate supplementation. In contrast, sodium acetate failed to restore EPS synthesis in the Xcc madB mutant. Biochemical and genetic analyses indicated that these divergent physiological roles arise from the distinct biochemical functions of MadB in the two bacteria. In Xoo , the fatty acid synthesis (FAS) pathway mediated by MadB operates independently of the FAS pathway mediated by FabH. Conversely, in Xcc , the FAS pathway mediated by FabH is the primary route, with MadB’s pathway serving a supplementary and regulatory role. Further analysis of gene organization and expression regulation of madB in both bacteria corroborates these distinctions. IMPORTANCE Despite the high conservation of the mad gene within the Proteobacteria, the physiological roles of the Mad protein remain largely unclear. Xoo and Xcc are bacteria with very close phylogenetic relationships, both encoding malonyl-ACP decarboxylase (MadB). However, MadB demonstrates substantial physiological function variations between these two species. This study demonstrates that even in closely related bacteria, homologous genes have adopted different evolutionary pathways to adapt to diverse living environments, forming unique gene expression regulation mechanisms. This has led to the biochemical functional divergence of homologous proteins within their respective species, ultimately resulting in distinct physiological functions.