Previously, we identified valosin-containing protein (VCP) as a mediator of ER stress-induced cell death. Mutations in the VCP gene including R93, R155, and R191 have been described that manifest clinically as hereditary inclusion body myopathy with Paget's disease of bone and frontotemporal dementia. In addition, other studies have demonstrated that as a consequence of a mutation generated in the second ATP binding domain of VCP (K524A), cells accumulated large cytoplasmic vacuoles and underwent programmed cell death. In order to better understand the biochemical and molecular consequences of the clinically relevant VCP mutations as well as the genetically engineered ATPase-inactive mutant K524A and any relationship these may have to ER stress-induced cell death, we introduced analogous mutations separately and together into the human VCP gene and evaluated their effect on proteasome activity, Huntingtin protein aggregation and ER stress-induced cell death. Our results indicate that the VCP K524A mutant and the triple mutant VCP R93C-R155C-K524A block protein degradation, trigger Huntingtin aggregate formation, and render cells highly susceptible to ER stress-induced cell death as compared to VCPWT or other VCP mutants.