BACKGROUND AND PURPOSE: Dynasore has been used extensively as an inhibitor of clathrin-mediated endocytosis. While studying the role of endocytosis in LPS-induced signalling events, we discovered that dynasore itself induced activation of NF-kappaB, independently of its effects on endocytosis and without involving the Toll-like receptor 4 signalling pathways. The purpose of this study was to characterize this novel effect and to explore the underlying mechanism of action. EXPERIMENTAL APPROACH: We utilized gel electrophoresis, microscopy, gene knockdown and luciferase-based promoter activity to evaluate the effect of dynasore on cell signalling pathways and to delineate the mechanisms involved in its effects, KEY RESULTS: Dynasore activated the NF-kappaB and IFN-beta pathways by activating mitochondrial antiviral signalling protein (MAVS). We showed that MAVS is activated by NOX/Rac and forms high molecular weight aggregates, similar to that observed in response to viral infection. We also demonstrated that dynasore-induced activation of JNK occurs downstream of MAVS and is required for activation of NF-kappaB and IFN-beta. CONCLUSION AND IMPLICATIONS: These findings demonstrate a novel effect of dynasore on cell signalling. We describe a novel Rac1-, ROS- and MAVS-mediated signalling cascade through which dynasore dramatically activates NF-kappaB, mimicking the viral induction of this key inflammatory signalling pathway. Our results call attention to the need for a broader interpretation of results when dynasore is used in its traditional fashion as an inhibitor of clathrin-mediated endocytosis. These results suggest the intriguing possibility that dynasore or one of its analogues might be of value as an antiviral therapeutic strategy or vaccine adjuvant.