NKG2D activation plays an important role in initiating and maintaining liver inflammation, and blockade of NKG2D recognition becomes a promising approach to alleviate liver inflammation. Treatment by silencing NKG2D ligands on hepatocytes, but not NKG2D on circulating immune cells, is more liver‐specific, and simultaneous knockdown of multiple NKG2D ligands on hepatocytes will be more efficient in liver disease intervention. Here, we constructed a single vector that could simultaneously express multiple short hairpin RNAs (shRNAs) against all murine NKG2D ligands including Rae1, Mult1, and H60. After hydrodynamic injection of plasmid containing the three shRNA sequences (shRae1‐shMult1‐shH60), also called pRNAT‐shRMH, we found the expression of all three NKG2D ligands on hepatocytes was downregulated both on messenger RNA and protein levels. Moreover, natural killer (NK) cell–mediated NKG2D‐dependent fulminant hepatitis of the mice was alleviated, along with inactivation of hepatic NK cells, by pRNAT‐shRMH if compared with its counterpart RNA interference vectors against single or double ligands. The therapeutic efficacy of pRNAT‐shRMH was equivalent to that of injecting three monoclonal antibodies against Rae1, Mult1, and H60. For better in vivo application, we constructed a recombinant adenovirus containing pRNAT‐shRMH (called Ad‐RMH) with efficient hepatotropic infection capacity and observed that Ad‐RMH intravenous injection exerted a similar therapeutic efficiency as plasmid pRNAT‐shRMH hydrodynamic injection. Noticeably, simultaneous knockdown of multiple human NKG2D ligands (MICA/B, ULBP2, and ULBP3) also significantly attenuated NK cell cytolysis against human NKG2D ligand‐positive hepatocyte L‐02 cells, suggesting a possible translation into human settings. Conclusion: Simultaneous knockdown of multiple ligands of NKG2D prevents NK cell–mediated fulminant hepatitis and is a potential therapeutic approach to treat liver diseases. (HEPATOLOGY 2013)