AbstractElizabethkingia miricola is a multidrug-resistant pathogen that can cause life-threatening infections in immunocompromised humans and outbreaks in amphibians. However, the specific virulence factors of this microorganism have not been described. In this study, we identified the polysaccharide biosynthesis protein-encoding gene capD, which is located in the conserved region of the Wzy-dependent capsule synthesis gene cluster in the E. miricola strain FL160902, and investigated its role in the pathogenesis of E. miricola. Our results revealed that the capD deletion strain (ΔcapD) lost its typical encapsulated structure, with a 45% reduction in cell wall thickness. CapD affects wza expression in the capsule polysaccharide synthesis pathway. Furthermore, the survival rates were significantly reduced in ΔcapD in response to complement-mediated killing, desiccation stress, and macrophage phagocytosis, whereas biofilm formation, surface hydrophobicity, and adherence to both endothelial and epithelial cells were increased. Additionally, the deletion of capD sharply attenuated the virulence of E. miricola in a frog infection model. Complementation of the capD gene restored the biological properties and virulence to wild-type levels. Overall, these findings suggest that CapD contributes to polysaccharide synthesis and plays a crucial role in the pathogenesis of E. miricola.