Human leukocyte antigen-G (HLA-G) is a nonclassical tolerogenic molecule that can be expressed either as membrane bound (HLA-G1) or secreted (HLA-G5) isoforms. Upregulation of HLA-G1 or HLA-G5 expression by tumor cells constitutes an efficient way to escape from antitumoral immune responses. The inhibitory role of HLA-G1 on NK cell cytotoxicity is well characterized; however, that of the HLA-G5 isoform secreted by tumor is poorly understood. Our results indicate that the HLA-G5 isoform secreted by M8 melanoma cells is able to protect them from natural killer leukemia cell line (NKL) cytotoxicity. Analysis of NKL/M8-HLA-G5 conjugates by confocal microscopy demonstrates that the inhibition of NKL cytotoxic activity resulted from an impairment of NKL actin reorganization and perforin granules polarization toward M8-HLA-G5 target cell. This study also indicates that HLA-G5 soluble isoform remains evenly distributed in the cytoplasm of M8-HLA-G5 conjugated to NKL cells, suggesting that HLA-G5 does not require to polarize toward effector cell to induce efficient inhibition. These results highlight the inhibitory mechanisms mediated through HLA-G5 leading to tumor escape from NK cell cytotoxicity.