Objective: To provide a kinetic model(s) and reveal the mechanism of thymoquinone and Poloxin blocking an emerging anti-cancer target, human Polo-like kinase 1 (hPlk1) Polo-box domain (PBD). Methods: The binding kinetics was determined by using a fluorescence polarization based assay. The putative mechanism was examined with a competition test. Results: Thymoquinone follows a one-step binding with an association rate constant (k1) of 6.635×103 L·mol-1·min-1. Poloxin fit a two-step binding with a dissociation constant (Ki) of 118 μmol/L for the intermediate complex and its isomerization rate (k4) of 0.131 5 min-1 to form an irreversible adduct. No significant dissociation was observed for either ligand up to 13 h. The inhibitors responded insignificantly to the presence of Michael donors as hPlk1-PBD competitors. Conclusion: Thymoquinone and Poloxin are slow-tight ligands to the hPlk1-PBD with kinetic models distinct from each other. Michael addition as the mechanism is excluded.