We previously demonstrated that the flagellin of intracellularVibrio splendidusAJ01 could be specifically identified by tropomodulin (Tmod) and further mediate p53-dependent coelomocyte apoptosis in the sea cucumberApostichopus japonicus. In higher animals, Tmod serves as a regulator in stabilizing the actin cytoskeleton. However, the mechanism on how AJ01 breaks the AjTmod-stabilized cytoskeleton for internalization remains unclear. Here, we identified a novel AJ01 Type III secretion system (T3SS) effector of leucine-rich repeat-containing serine/threonine-protein kinase (STPKLRR) with five LRR domains and a serine/threonine kinase (STYKc) domain, which could specifically interact with tropomodulin domain of AjTmod. Furthermore, we found that STPKLRR directly phosphorylated AjTmod at serine 52 (S52) to reduce the binding stability between AjTmod and actin. After AjTmod dissociated from actin, the F-actin/G-actin ratio decreased to induce cytoskeletal rearrangement, which in turn promoted the internalization of AJ01. The STPKLRR knocked out strain could not phosphorylated AjTmod and displayed lower internalization capacity and pathogenic effect compared to AJ01. Overall, we demonstrated for the first time that the T3SS effector STPKLRR with kinase activity was a novel virulence factor inVibrioand mediated self-internalization by targeting host AjTmod phosphorylation dependent cytoskeleton rearrangement, which provided a candidate target to control AJ01 infection in practice.