ABSTRACT: Hand, foot, and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified as having potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all EV71, CVA16, and CVA6 strains tested. In rhabdomyosarcoma cells, the 50% inhibitory concentrations of the dye E151 for various strains of EV71 ranged from 2.39??M to 28.12??M, whereas its 50% cytotoxic concentration was 1,870??M. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy in viral inhibition was regulated by amino acids at VP1-98, VP1-145, and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted attached viruses in a concentration-dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200?mg/kg of body weight/day given on the initial 4 days of challenge protected AG129 mice challenged with 10× the 50% lethal dose of wild-type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection.IMPORTANCE Human enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease in children and is responsible for thousands of deaths in the past 20?years. Food azo dyes have been widely used since the nineteenth century; however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered that one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent.