Valosin-containing protein (VCP or p97) is required for the proteasomal degradation of polyubiquitinated proteins. However, the molecular mechanism for VCP to process the polyubiquitinated proteins remains unclear. Here, we show that VCP can unfold polyubiquitinated proteins. It preferably unfolds the pentaubiquitin-over monoubiquin-conjugated dihydrofolate reductase (Ub5-DHFR or Ub-DHFR) in a dose dependent manner. In addition, the unfolding activity of VCP does not depend on its ATPase activity, on the contrary, ATP and its non-hydrolysable analogs suppress the unfolding of Ub5-DHFR. The structural and functional analysis showed that either D1 or D2 domain of VCP is sufficient to carry out this unfolding activity. The structure of the substrates also affects its unfolding by VCP. VCP is unable to unfold Ub5-DHFR in a tight structure when it binds with methotrexate, a folate analog with high affinity to DHFR. Thus, these results support that VCP is capable of unfolding polyubiquitinated proteins and suggest that VCP may facilitate the proteasomal degradation of polyubiquitinated proteins through its unfolding activity. (C) 2015 Elsevier Inc. All rights reserved.