Regional recruitment of dendritic cells (DCs) by the local administration of granulocyte macrophage-colony stimulating factor (GM-CSF) or Flt3-ligand (Flt3L) has vaccine adjuvant activity. However, Flt3L, with its DC growth factor activity, has not been extensively studied as a vaccine adjuvant, particularly as a plasmid vector. We report that the intramuscular (IM) injection of a Flt3L plasmid (pNGVL-hFlex), when formulated in a pluronic carrier (SP1017, Supratek Pharma, Inc., Laval, Que., Canada), recruits DC to the injection site and regional lymph nodes (LNs) and augments immune responses to a p17 HIV plasmid vaccine to a greater extent than the injection of a naked DNA vaccine alone. Following IM administration of pNGVL-hFlex, Flt3L mRNA, Flt3L protein and infiltrating DC accumulate at the injection site. The number of DC in the draining LNs are also significantly increased with the greatest increase observed following injection of 2.5μg of pNGVL-hFlex formulated in 0.01% SP1017. Flow cytometric studies demonstrate that the LN-infiltrating DC is mainly of the CD11c⁺CD11b⁻ phenotype (IL-12 producing). Further, the co-injection of pNGVL3-hFlex and p17 HIV plasmids, formulated in SP1017, significantly increases the immune responses to the plasmid vaccine (pVAX-gag). The co-injection of pVAX-gag and pNGVL3-hFlex, formulated in SP1017, significantly increase delayed-type hypersensitivity responses and the numbers of antigen (Ag)-specific interferon-γ secreting T cells in the spleen (Enzyme Linked Immune Spot (ELISpot) assay), compared to mice immunized with pVAX-gag formulated in SP1017 alone. We conclude that the IM injection of pNGVL-hFlex with SP1017 can increase the number of DC in draining LN and at the site of injection, thereby providing adjuvant activity for a plasmid vaccine resulting in a significantly increased, Ag-specific T cell response.