As sexual transmission of human immunodeficiency virus-1 (HIV-1) occurs via the mucosa, an ideal HIV-1 vaccine should induce both mucosal and systemic immunity. We therefore sought to evaluate the induction of mucosal responses using a DNA env prime-gp120 protein boost approach in which sequential nasal and parenteral protein administration was performed with two novel carbohydrate-based adjuvants. These adjuvants, Advax-M and Advax-P, were specifically designed for mucosal and systemic immune enhancement, respectively. Murine intranasal immunization with gp120/Advax-M adjuvant elicited gp120-specific IgA in serum and mucosal secretions that was markedly enhanced by DNA priming. Boosting of DNA-primed mice with gp120/Advax-M and gp120/Advax-P by sequential intranasal and intramuscular immunization, or vice versa, elicited persistent mucosal gp120-specific IgA, systemic IgG and memory T- and B-cell responses. Induction of homologous, but not heterologous, neutralizing activity was noted in the sera of all immunized groups. While confirmation of efficacy is required in challenge studies using non-human primates, these results suggest that the combination of DNA priming with sequential nasal and parenteral protein boosting, with appropriate mucosal and systemic adjuvants, could generate strong mucosal and systemic immunity and may block HIV-1 mucosal transmission and infection.