Persistent B cell responses in mucosal tissues are crucial to control infection against sexually transmitted pathogens like human immunodeficiency virus 1 (HIV-1). The genital tract is a major site of infection by HIV. Sublingual (SL) immunization in mice was previously shown to generate HIV-specific B cell immunity that disseminates to the genital tract. We report here the immunogenicity in female cynomolgus macaques of a SL vaccine based on a modified gp41 polypeptide coupled to the cholera toxin B subunit designed to expose hidden epitopes and to improve mucosal retention. Combined SL/intramuscular (IM) immunization with such mucoadhesive gp41-based vaccine elicited mucosal HIV-specific IgG and IgA antibodies more efficiently than IM immunization alone. This strategy increased the number and duration of gp41-specific IgA secreting cells. Importantly, combined immunization improved the generation of functional antibodies 3months after vaccination as detected in HIV-neutralizing assays. Therefore, SL immunization represents a promising vaccine strategy to block HIV-1 transmission.