CTB-MPR649-684, a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649-684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB-MPR649-684 was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB-MPR649-684 were equivalent to their nanomolar affinities toward an MPR peptide. The fusion proteins affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR649-684 raised only a modest level of anti-MPR649-684 Abs. However, a prime-boost immunization with CTB-MPR649-684 and a second MPR649-684-based immunogen elicited a more productive anti-MPR649-684 antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells – Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPRs N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR649-684-based immunogens may provide broad protective value independent of infection neutralization.