Introduction:Streptococcus suis(S. suis) is an important swine pathogen that cause a wide range of diseases and is also a occasionally pathogen for humans leading life-threatening diseases.A number of studies have shown that Serine/Threonine Protein Kinase (STK) is commonly found in bacteria and has a diversity of STK substrates involving in important life processes of bacteria, including cell division and morphology.Studies from our and other groups have shown that inactivation of stk greatly affected normal cell division of S. suis, however, there is no report on the phosphorylated substrate of STK in S. suis. Objective: Identification of S. suis STK phosphorylated substrate proteins, and getting insight into the underline mechanism. Results: Seven differentially phosphorylated proteins were identified by phosphoproteomicsan alysis for wild type and stk gene knock out S. suis strains. One of them was annotated as cell division initiation protein (DivIVA), which have been reported to regulate cell division in several bacteria. Western analysis using an anti-threonine phosphorylation antibody revealed that the recombinant STK protein could successfully phosphorylate DivIVAprotein in vitro. To determine the specific amino acid residue that phosphorylated by STK, mass spectrometry was performed, which identified a threonineat position 199 of DivIVAas a potential phosphorylation site. In support of this finding, replacing threonineat position 199 of DivIVA with analanine completely abolished its phosphorylation by STK.Conclusion:Both in vivo and in vitro experiments support that DivIVAis aphosphorylated substrate of STK in S. suis.The results suggest that STK may regulate cell division in S. suis through modulating DivIVA phosphorylation.