Inhibitors of human p97 (also known as valosin-containing protein) have been actively pursued because of their potential therapeutic applications in cancer and other diseases. However, covalent and irreversible p97 inhibitors have not been well explored. Herein, we report our design, synthesis, and biological evaluation of covalent and irreversible inhibitors of p97. Among an amide and a reverse amide series we synthesized, we have identified a p97 inhibitor whose functional irreversibility has been established both in vitro and in cells. Also importantly, mass spectrometry reveals three potential cysteine residues labeled by this compound, and mutagenesis together with computer modeling suggests Cys522 as a major site, which when modified, could compromise the function of p97. Taken together, this new inhibitor may provide a template for designing more potent p97 inhibitors with covalent and irreversible characteristics.