Xanthoxyletin　(C15H14O4),　a　natural　linear　pyranocoumarin,　is　known　to　trigger　anti-cancer　activities,　although　its　anticancer　mechanisms　are　largely　unknown.　Human　hemoglobin　(HHb)　as　a　major　plasma　protein　could　play　a　key　role　in　the　transportation　and　biodistribution　of　therapeutic　molecules　In　this　study,　the　interaction　of　xanthoxyletin　with　HHb　was　measured　under　physiological　conditions　using　multi-spectroscopic　techniques　along　with　molecular　docking　simulation.　Also,　the　interaction　of　xanthoxyletin　with　human　chronic　myeloid　leukemia　K562　cells　was　assessed　by　cellular　assays.　It　was　deduced　that　each　xanthoxyletin　molecule　shows　a　thermodynamically　favorable　interaction　with　one　molecule　of　HHb　with　a　binding　constant　(Kb)　in　the　orders　of　103-4　M−1.　These　interactions　did　not　significantly　result　in　the　loss　of　α-helical　content　of　HHb.　In　silico　studies　supported　the　experimental　findings　and　demonstrated　that　non-covalent　forces　including　hydrogen　bonds　(Ala88　and　Arg92),　pi-pi　stacking　(Trp37),　and　hydrophobic　interactions　(and　Pro95　and　Pro36)　are　the　main　forces　contributing　to　the　xanthoxyletin-HHb　complex　formation.　Cellular　and　molecular　assays　showed　that　xanthoxyletin　mitigated　the　proliferation　of　K562　cells　through　apoptosis　induction　mediated　by　overexpression　of　p53,　caspase-3,　and　PARP　cleavage.　This　data　may　hold　great　promise　for　the　development　of　anticancer　compounds　with　potential　blood　protein　binding　stemming　from　xanthoxyletin　derivatives/hybrids.　©　2024　The　Authors