Direct DNA inoculation can induce both protective humoral and cellular responses against several viruses. The HTLV-I envelope glycoproteins are the major antigens recognized by sera of HTLV-I infected patients that generate neutralizing immune responses in vitro and in vivo. We compared immune responses elicited after a single inoculation of two plasmids encoding the complete HTLV-I envelope proteins followed or not by gp62 Baculovirus recombinant protein boosts in BALB/c mice. First, we observe that the coexpression of env and rex genes is not sufficient to raise a detectable specific humoral response after a single DNA inoculation. Protein boosts generated a high antibody response in mice primed with DNA expressing HTLV-I envelope proteins as compared to naive and negative control vector primed groups. This humoral response presented high neutralizing antibody titers. These results suggest that a single inoculation of DNA expressing HTLV-I env gene can stimulate memory B-cell clones that are able to respond effectively to subsequent encounters with HTLV-I envelope proteins and a specific cellular T helper cell response in mice.