Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. citri, car A and car B encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that car A did not affect any of the phenotypes, while car B played multiple roles in pathogenicity. The deletion of car B rendered the loss of pathogenicity in host plants and the ability to induce a hypersensitive reaction in the non-hosts. Quantitative reverse transcription-PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in car B also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data demonstrated that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri. Carbamoyl-phosphate synthase plays a vital role in the carbon and nitrogen metabolism cycles. In Xanthomonas citri subsp. Citri, car A and car B encode the small and large subunits of carbamoyl-phosphate synthase, respectively. The deletion mutation of the coding regions revealed that car A did not affect any of the phenotypes, while car B played multiple roles in pathogenicity. The deletion of car B rendered the loss of pathogenicity in host plants and the ability to induce hypersensitive reaction in the non-hosts. Quantitative reverse transcription -PCR assays indicated that 11 hrp genes coding the type III secretion system were suppressed when interacting with citrus plants. The mutation in car B also affected bacterial utilization of several carbon and nitrogen resources in minimal medium MMX and extracellular enzyme activities. These data are that that only the large subunit of carbamoyl-phosphate synthase was essential for canker development by X. citri subsp. citri.