Granzyme K (GzmK) highly expressed in NK and NKT cells. We recently demonstrated that GzmK induces rapid caspase-independent cell death with ssDNA nicks. Little is known about its molecular mechanisms to mediate caspase-independent cell death. In this study, we found the valosin-containing protein (VCP) is a physiological substrate of GzmK. GzmK cleaves VCP at residue Arg(713) in the D2 domain and abrogates its ATPase activity. GzmK can also target other endoplasmic reticulum-associated degradation complex components Ufd1 and Npl4. Disruption of the endoplasmic reticulum-associated degradation pathway after GzmK treatment initiates ubiquitinated protein accumulation leading to xbp1 splicing. These indicate that ubiquitinated protein accumulation triggers endoplasmic reticulum stress in target cells. In support of this, target tumor cells with silenced VCP expression are more sensitive, whereas cells overexpressing VCP are more resistant to GzmK-mediated cytotoxicity.