Plasma protein binding of therapeutic bioactive materials, especially anticancer compounds can play an important role in their pharmacokinetics and pharmacodynamics. Human alpha-1 acid glycoprotein (AGP) has shown potential binding affinities to different endogenous and exogenous ligands due to its unique binding site composed of eight anti-parallel beta-sheets. Therefore, in this study, the interaction of nordentatin, a member of coumarins with a chemical formula of C(19)H(20)O4 with AGP, was measured by multi-spectroscopic and theoretical approaches. Afterward, the anticancer potency of nordentatin against human colorectal cancer HCT-116 cells was assessed. The fluorescence quenching of AGP was observed upon binding of nordentatin through a static quenching mechanism. The spontaneous hydrophobic interaction of nordentatin with AGP had a binding constant value (K-b) of 10(4) M-1 with a marginal change in the secondary structure of AGP. Cellular assays demonstrated that nordentatin exhibited promising inhibitory effects on the cellular growth of HCT-116 cells through increasing the levels of intracellular ROS and [Ca2+], while it remarkably triggered the MMP reduction. Also, RT-qPCR analysis disclosed that nordentatin stimulated apoptosis by overexpression of P53, Bax/Bcl-2, cytochrome c, caspase-9, and caspase-3 mRNA. Overall, these findings suggested that nordentatin, in addition to potential binding to plasma protein, could be used as an effective preventive or curative plant-based therapy for colorectal cancer.