Background: Nasopharyngeal cancer is a tumor that occurs in the mucous epithelium of the nasopharynx. Due to its rapid growth and early metastatic nature, the successful treatment of nasopharyngeal cancer is highly challenging.Objective: Here, we intended to assess the in vitro anticancer property of brassinin against the nasopharyngeal cancer C666-1 cells.Methodology: The in vitro free radical scavenging property of the brassinin was assessed by var-ious free radical scavenging activities such as FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The cytotoxic level of the brassinin (1-50 mM) against the nasopharyngeal cancer C666-1 cells and normal Vero cells were assessed by the MTT cytotoxicity assay. The levels of TBARS, GSH, and the SOD activity was assessed using kits. The level of ROS generation, MMP, and apop-tosis were investigated by the respective fluorescent staining techniques. The flow cytometry analysis was done to scrutinize the cell cycle arrest. The Bax/Bcl-2 level and caspase activities were examined using respective kits.Results: The brassinin treatment effectively scavenged the free radicals, which are assessed by the FRAP, DPPH, chemiluminescence (CL), and ORAC assays. The proliferation of brassinin treated C666-1 cells were decreased remarkably, while the same concentration of brassinin did not dis-turbed the Vero cell viability. The 30 mM of brassinin effectively increased the ROS production, depleted the MMP, and stimulated the apoptosis in the C666-1 cells. The brassinin increased the TBARS and depleted the GSH and SOD in the C666-1 cells. The flow cytometry analysis revealed that the brassinin administration improved the G0/G1 ratio and decreased the proportion of cells with 'S' and 'G2/M' phase. The Bax, caspase-3 and -9 were elevated and Bcl-2 level was decreased in the brassinin administered C666-1 cells.Conclusion: Our findings discovered that the brassinin has the capacity to prevent the prolifer-ation and stimulate the apoptotic cell death C666-1 cells via blocking cell cycle and increasing oxidative stress and apoptotic markers. Hence, it can be a talented therapeutic agent to treat the nasopharyngeal cancer in the future. Crown Copyright (c) 2022 Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).