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High Mobility Group protein 1 (HMGB1) is a non-histone, chromatin-associated nuclear protein that functions in regulating eukaryotic gene expression. We investigated the influence and mechanism of action of Aedes aegypti HMGB1 (AaHMGB1) on mosquito Rel1A-mediated transcription from target gene promoters. The DNA-binding domain (RHD) of AaRel1A was bacterially expressed and purified, and AaHMGB1 dramatically enhanced RHD binding to consensus NF-kB/Rel DNA response elements. Luciferase reporter analyses using a cecropin gene promoter showed that AaHMGB1 potentiates the transcriptional activity of AaRel1A in Aag-2 cells. Moreover, overexpression of AaHMGB1 in Aag-2 cells led to an increase in mRNA levels of antimicrobial peptide genes. In vitro GST pull-down assays revealed that the presence of DNA is a pre-requisite for assembly of a possible ternary complex containing DNA, AaHMGB1 and AaRel1A. Notably, DNA bending by AaHMGB1 enhanced the binding of AaRel1A to a DNA fragment containing a putative NF-kB/Rel response element. Importantly, AaHMGB1 was identified as a potential immune modulator in A. aegypti through AaHMGB1 overexpression or RNAi silencing in Aag-2 cells followed by bacterial challenge or through AaHMGB1 RNAi knockdown in mosquitoes followed by Dengue virus (DENV) infection. We propose a model in which AaHMGB1 bends NF-kB/Rel target DNA to recruit and allow more efficient AaRel1A binding to activate transcription of effector genes, culminating in a stronger Toll pathway-mediated response against DENV infection.