Riemerella anatipestifer (R. anatipestifer) is a major bacterial pathogen that causes pericarditis, perihepatitis, and airsacculitis in ducks. However, the pathogenesis of R. anatipestifer is still unclear. Type IX secretion system (T9SS) has been reported to be related to the pathogenic properties of the phylum Bacteroidetes, gldK gene was a major component of this secretion. In this study, we used a homologous recombination method to construct a gldK mutant strain, and found that the survival rate of gldK mutant strain infected ducks was 80%, while R. anatipestifer CH-1 wild-type-infected ducks was only 20%, the median lethal dose (LD50) of the gldK mutant strain was 2.25 × 1011, which is approximately 1.5 × 103-fold higher than that of the wild-type strain (1.44 × 108). The bacterial loads in the blood, liver, and brain tissues of gldK mutant strain-infected ducks were also considerably lower than those of R. anatipestifer CH-1 wild-type-infected ducks. In conclusion, gldK gene can be deleted, suggesting that the gldK gene was a non-essential gene for growth. However, it plays an important role in the bacterial virulence of R. anatipestifer CH-1.