Protein‐based vaccines offer safety and cost advantages but require adjuvants to induce immunity. Here we examined the adjuvant capacity of glucopyranosyl lipid A (GLA), a new synthetic non‐toxic analogue of lipopolysaccharide. In mice, in comparison with non‐formulated LPS and monophosphoryl lipid A, formulated GLA induced higher antibody titers and generated Type 1 T‐cell responses to HIV gag‐p24 protein in spleen and lymph nodes, which was dependent on TLR4 expression. Immunization was greatly improved by targeting HIV gag p24 to DCs with an antibody to DEC‐205, a DC receptor for antigen uptake and processing. Subcutaneous immunization induced antigen‐specific T‐cell responses in the intestinal lamina propria. Immunity did not develop in mice transiently depleted of DCs. To understand how GLA works, we studied DCs directly from vaccinated mice. Within 4 h, GLA caused DCs to upregulate CD86 and CD40 and produce cytokines including IL‐12p70 in vivo. Importantly, DCs removed from mice 4 h after vaccination became immunogenic, capable of inducing T‐cell immunity upon injection into naïve mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later.