To facilitate the development of p97 inhibitors as potential therapeutic agents and help define their clinical application, it is necessary to dissect the mechanism of action (MOA) of p97 inhibitors and compare them with proteasome inhibitors. Moreover, identifying specific cellular markers is critical for both the drug discovery and development process, to help validate candidate drugs and quantify their effect. In this study, we performed proteomic analysis and systematically compared p97 shRNA knockdown (KD), and three p97 inhibitors, with the proteasome inhibitor MG132 in HCT116 colon cancer cells. Through proteomic analyses we uncover the specific molecular mechanism by which p97 inhibition is distinct from proteasome inhibition, potentially relevant to understanding the efficacious effects of the former in solid tumors.