Rhytidenones family are spirobisnaphthalene natural products isolated from mangrove endophytic fungus Rhytidhysteron rufulum AS21B. In this study, we finished the biomimetic synthesis of complex rhytidenone A, and elucidated the mode of action of the highly cytotoxic rhytidenone F using chemical biology approach. Rhytidenone F covalently targets PA28γ by selectively binding to Cys92 residue and leads to the cellular accumulation of p53. Consequently, the Fas-dependent signalling pathway will be activated to initiate cellular apoptosis. Our studies have identified the first small molecule inhibitor targeting PA28γ, suggesting rhytidenone F may serve as a promising natural product lead for future anticancer drug development.