Hepatocyte growth factor (HGF) is involved in the pathogenesis of Kaposi's sarcoma (KS), the most frequent neoplasia in patients with AIDS, characterized by proliferating spindle cells, infiltrating inflammatory cells, angiogenesis, edema, and invasiveness. In vitro, this factor sustains the biological behavior of KS derived cells, after activation of its receptor and the downstream MAPK and AKT signals. In other cell types, namely endothelial and epithelial cells, movement, proliferation, and survival stimulated by HGF and other growth factors and cytokines depend on diacylglycerol kinases (DGK). In an effort to identify new intracellular transducers operative in KS cells, which could represent therapeutic targets, we investigated the role of DGK in KS cell movement and proliferation by treating cells with the DGK pharmacological inhibitor R59949. We report that R59949 strongly inhibits HGF-induced KS motility, proliferation, and anchorage-independent growth with only a partial effect on cell adhesion and spreading. R59949 does not affect cell survival, HGF receptor activation, or the classical MAPK and AKT signalling pathways. Furthermore, we carried out an siRNA screen to characterize the DGK isoforms involved in KS motility and anchorage independent growth. Our data indicate a strong involvement of DGK-δ in KS motility and of DGK-ι in anchorage-independent growth. These results indicate that DGK inhibition is sufficient to impair in vitro KS cell proliferation and movement and suggest that selected DGK represent new pharmacological targets to interfere with the malignant properties of KS, independently from the well-known RAS/MAPK and PI3K/AKT pathways.