BACKGROUND: Berberine (BBR), an isoquinoline alkaloid, has been applied clinically to treat dysentery caused by Shigella for decades. Nevertheless, the precise mechanisms behind its anti-Shigella effect have not been fully elucidated. PURPOSE: This study aims to investigate the mechanism of BBR on antibacterial activity against S. flexneri infection. METHODS: We initially reproduced the mouse model of Shigella flexneri-induced dysentery, and then, assessed the therapeutic effect of BBR. In vitro, we measured the inhibitory effect of BBR against S. flexneri and the GTPase activity of FtsZ (filamentous temperature sensitive protein Z) using the minimum inhibitory concentration (MIC) test and an enzyme activity assay to investigate the bacteria-directed mechanisms. Subsequently, we utilized both the in vivo mouse model of dysentery and the in vitro macrophage infection model with S. flexneri to explore the host-directed anti-Shigella mechanisms of BBR. The canonical pyroptosis pathway mediated by caspase-1 and mitochondrial damage were examined by Western blot, immunofluorescence and RNA interference analysis. RESULTS: Administration of BBR alleviated the symptoms of dysentery induced by S. flexneri infection. In vitro, BBR could inhibit the growth of S. flexneri by targeting the GTPase activity of FtsZ, thereby affecting bacterial cell division. Additionally, our in vivo findings revealed that BBR suppressed macrophage pyroptosis by inhibiting the expression of caspase-1 and subsequently the mitochondrial damage, which in turn reduced the intestinal inflammation and tissue damage. CONCLUSIONS: Our results provide a novel mechanism of BBR's action, which targets both the bacterium and the host to exert its antibacterial effects. Furthermore, it also provides an explanation for the discrepancy between BBR's relatively modest antibacterial efficacy in vitro and its enhanced antibacterial effects in vivo, thus, giving support to its clinical use.