Purpose: Hepatocellular carcinoma (HCC) displays particular resistance to conventional cytostatic agents. Alternative treatment strategies focus on novel substances exhibiting antineoplastic and/or immunomodulatory activity enhancing for example natural killer (NK) cell antitumor reactivity. However, tumor-associated ligands engaging activating NK cell receptors are largely unknown. Exceptions are NKG2D ligands (NKG2DL) of the MHC class I-related chain and UL16-binding protein families, which potently stimulate NK cell responses. We studied the consequences of proteasome inhibition with regard to direct and NK cell-mediated effects against HCC. Experimental Design: Primary human hepatocytes (PHH) from different donors, hepatoma cell lines, and NK cells were exposed to Bortezomib. Growth and viability of the different cells, and immunomodulatory effects including alterations of NKG2DL expression on hepatoma cells, specific induction of NK cell cytotoxicity and IFN-gamma production were investigated. Results: Bortezomib treatment inhibited hepatoma cell growth with IC50 values between 2.4 and 7.7 nmol/L. These low doses increased MICA/B mRNA levels, resulting in an increase of total and cell surface protein expression in hepatoma cells, thus stimulating cytotoxicity and IFN-gamma production of cocultured NK cells. Importantly, although NK cell IFN-gamma production was concentration-dependently reduced, low-dose Bortezomib neither induced NKG2DL expression or cell death in PHH nor altered NK cell cytotoxicity. Conclusions: Low-dose Bortezomib mediates a specific dual antitumor effect in HCC by inhibiting tumor cell proliferation and priming hepatoma cells for NK cell antitumor reactivity. Our data suggest that patients with HCC may benefit from Bortezomib treatment combined with immunotherapeutic approaches such as adoptive NK cell transfer taking advantage of enhanced NKG2D-mediated antitumor immunity.