Background HIV-1 sexual transmission occurs mostly through infected semen, which contains both free virions and infected leukocytes. Transmission initiated by infected cells has been shown by several in vitro and in vivo studies and a reduced capacity of broadly neutralizing antibodies (bNAbs) to inhibit cell-to-cell transmission has also been reported. However, due to limitations of available experimental models, there is yet no clarity to which extend bNAbs can prevent transmission mediated by semen leukocytes. Methods We developed a novel in vitro assay to measure cell-cell transmission that makes use of splenocytes or CD45+ semen leukocytes collected from acutely SHIV162P3-infected cynomolgus macaques. A panel of 11 bNAbs was used either alone or in combination to assess their inhibitory potential against both cell-free and cell-cell infection. Findings Splenocytes and semen leucocytes displayed a similar proportion of CD4+ T-cell subsets. Either cell type transferred infection in vitro to target TZM-bl cells and PBMCs. Moreover, infection of macaques was achieved following intravaginal challenge with splenocytes. The anti-N-glycans/V3 loop bNAb 10–1074 was highly efficient against cell-associated transmission mediated by infected spleen cells and its potency was maintained when transmission was mediated by CD45+ semen leukocytes. Interpretation These results support the use of bNAbs in preventative or therapeutic studies aiming to block transmission events mediated not only by free viral particles but also by infected cells. Our experimental system could be used to predict in vivo efficacy of bNAbs.