The formation of biofilm by pathogen promotes its colonization, posing significant threats to the environment and the health of humans and animals. The mechanism by which c-di-GMP regulates biofilm formation and its impact on pathogen colonization and intestinal injury has not been fully characterized. Here, we found that overexpressing the phosphodiesterases encoding gene adrB in Hafnia paralvei Z11 reduced c-di-GMP levels, leading to a decrease in outer membrane proteins (OMPs) content and the declining adhesion, as well as a lower level of biofilm formation. Conversely, overexpressing the OMPs-encoding gene lapA significantly enhanced adhesion and biofilm formation. Furthermore, overexpression of adrB exhibited a lower colonization ability in the loach intestine, whereas overexpression of lapA in strain Z11::pBBR1MCS-adrB significantly promoted its colonization in the loach intestine, and caused damage to intestinal barrier integrity, inducing host oxidative stress and the elevated expression level of inflammation-related genes. Thus, this study reveals that c-di-GMP regulates lapA-mediated biofilm formation of H. paralvei Z11, which promotes its intestinal colonization and damage to loach intestines, enhancing our understanding of pathogen-induced intestinal colonization and damage strategies.