The intracellular pathogen has evolved sophisticated mechanisms to evade host immune defenses by secreting different virulence factors. In our previous study, the eukaryotic factor STPKLRR was identified from the intracellular pathogen Vibrio splendidus AJ01 and shown to facilitate promote AJ01 internalization by mediating actin-dependent coelomocytes phagocytosis. However, the molecular mechanisms underlying AJ01'escaped from the phagosome remained largely unclear. In this study, a novel eukaryotic-like factor was identified, containing both the Serine/Threonine/Tyrosine (STYKc) domain and protein phosphatase 2 C (PP2C) domain (denoted as Sppsk1), which was essential for AJ01 phagosome escape. Deletion of Sppsk1 significantly increased phagolysosome maturation and reduced the intracellular AJ01 levels compared to the wild AJ01. Mechanistic analysis showed that the STYKc domain of Sppsk1 directly phosphorylated phagosome H