Licochalcone B (Lico B), which belongs to the retrochalcone family, is isolated from the roots of Chinese licorice. Lico B has been reported to have several other useful pharmacological properties, such as anti-inflammatory, antibacterial, antioxidant, antiulcer, anticancer, and anti-metastasis activities. We elucidated the underlying mechanism by which Lico B can induce apoptosis in oral squamous cell carcinoma (OSCC). Our results showed that exposure of OSCC cells (HN22 and HSC4) to Lico B significantly inhibited cell proliferation in a time- and concentration-dependent manner. Lico B caused cell cycle arrest at G1 phase along with downregulation of cyclin D1 and upregulation of p21 and p27 proteins. Lico B also facilitated the diffusion of phospholipid phosphatidylserine (PS) from inner to outer leaflets of the plasma membrane with chromatin condensation, DNA fragmentation, accumulated sub-G1 population in a concentration-dependent manner. Moreover, Lico B promoted the generation of reactive oxygen species (ROS), which, in turn, can induce CHOP, death receptor (DR) 4 and DR5. Lico B treatment induced downregulation of anti-apoptotic proteins (Bid and Bcl-xl and Mcl-1), and up-regulation of pro-apoptotic protein (Bax). Lico B also led to the loss of mitochondrial membrane potential (MMP), resulting in cytochrome c release. As can be expected from the above results, the apoptotic protease activating factor-1 (Apaf-1) and survivin were oppositely expressed in favor of apoptotic cell death. This notion was supported by the fact that Lico B activated multi-caspases with cleavage of poly (ADP-ribose) polymerase (PARP) protein. Therefore, it is suggested that Lico B is a promising drug for the treatment of human oral cancer via the induction of apoptotic cell death.