Pseudomonas donghuensis HYS is highly virulent to Caenorhabditis elegans, but with mechanistic details that are not fully understood. The hmuSTUV operon was reported to participate in the synthesis of heme in Pseudomonas. However, the exact role of the hmuSTUV operon in Pseudomonas virulence has not been elucidated. In this study, we report for the first time that the hmuSTUV operon in P. donghuensis HYS causes host virulence, and that hmuS was a key gene for the toxicity of this operon. Furthermore, RNA-seq data showed that hmuS deletion inhibited alginate gene expression, thereby inhibiting biofilm formation. The hmuSTUV operon and alginate gene cluster are conserved in Pseudomonas. By constructing mutant strains carrying GFP, we found that the hmuS deletion reduced colonisation of HYS to the host gut. Moreover, the expression of the alginate gene cluster was controlled by the construction of a L-arabinose-inducible promoter. hmuS positively regulated alginate gene cluster expression, mediating bacterial virulence against C. elegans. In addition, HYS originating from the East Lake of Wuhan City was more pathogenic to zebrafish than any other pathogenic Pseudomonas, through impairment of zebrafish neurodevelopment and locomotor ability, by colonizing to the zebrafish brain. In conclusion, the hmuSTUV operon positively regulated the alg gene cluster, thereby disabling bacterial biofilm formation and colonisation to mediate bacterial pathogenicity to the host. These novel findings revealed the critical interaction between the hmuSTUV operon and the alg gene cluster in the bacterial virulence of Pseudomonas, providing new insights into Pseudomonas pathogenicity.