Aim Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cardiocerebrovascular physiopathological process.It is also a promising anticancer target.It is highly desirable to discover novel NAMPT inhibitors as anticancer drug candidates and understand their action mode.Methods We carried out a high throughput screening system on a chemical library of 24434 smallmolecules.Antiproliferative activity were further studied on active compounds.Isothermal titration calorimetry and cellular thermal shift assay were used to confirm the target specificity.Molecular modeling and sitedirected mutagenesis studies were taken to investigate the binding mode of NAMPT inhibitor.Results Using high throughput screening system targeting NAMPT, we obtained a potent NAMPT inhibitor MS0 (China Patent ZL201110447488.9) with excellent in vitro activity (IC50 =9.87+ 1.15 nmol · L1) and antiproliferative activity against multiple human cancer cell lines including stemlike cancer cells.Structureactivity relationship studies yielded several highly effective analogues.These inhibitors specifically bound NAMPT, rather than downstream NMNAT.We provided the first chemical case using cellular thermal shift assay to explain the difference between in vitro and cellular activity; MS7 showed best in vitro activity(IC50 =0.93 + 0.29 nmol · L1) but worst cellular activity due to poor target engagement in living cells.Sitedirected mutagenesis studies identified important residues for NAMPT catalytic activity and inhibitor binding.Conclusions The present study provides a class of novel NAMPT inhibitors for future development of anticancer agents.Our findings also contribute to deep understanding the action mode of NAMPT inhibitors and NAMPT basic research in cardiocerebrovascular system.