Dengue virus (DENV) and other flaviviruses are prevented from replicating in mosquitoes by Wolbachia. To date, several reports have appeared that highlight multiple molecular and cellular pathways involved in the blocking mechanism, which underlines the complicated nature of the mechanism. Here, we developed a hypothesis on whether Wolbachia proteins interact with pro-viral host proteins by using a unique approach to study the antiviral mechanism based on Wolbachia-host protein-protein interaction. We selected Wolbachia surface protein (WSP) for co-immunoprecipitation tests because of its abundance and possible secretion. We first confirmed WSP’s secretion from mosquito cells and found two host proteins, Ae. aegypti Serine Threonine kinase (STK) and synaptic vesicle membrane protein VAT-1 (SVM) interacting with WSP. We examined the role of STK and SVM genes in relation to DENV replication in Ae. aegypti mosquitoes and mosquito cell lines with and without Wolbachia (Aag2 and Aag2.wAlbB). In DENV-infected Aag2 cells, expression studies revealed a substantial overexpression of SVM and STK. However, these genes were also induced in mock Aag2.wAlbB cells but downregulated after DENV infection. Silencing of STK, but not SVM, led to reduced DENV replication in Aag2 cells and mosquitoes, and RNA activation of STK, by utilising promoter induction via short activating oligos, resulted in higher DENV replication in Aag2 as well as Aag2.wAlbB cell lines. Overall, our findings suggest that STK serves as a pro-viral gene and Wolbachia WSP protein binds to it, possibly making it less accessible for DENV replication.