The isoquinoline alkaloid berberine possesses many pharmacological activities including antibacterial infection. Although the direct bactericidal effect of berberine has been documented, its influence on the antibacterial functions of macrophages is largely unknown. As inflammasome activation in macrophages is important for the defense against bacterial infection, we aimed to investigate the influence of berberine on inflammasome activation in murine macrophages. Our results showed that berberine significantly increased ATP-induced inflammasome activation as reflected by enhanced pyroptosis as well as increased release of caspase-1p10 and mature interleukin-1 beta (IL-1 beta) in macrophages. Such effects of berberine could be suppressed by AMP-activated protein kinase (AMPK) inhibitor compound C or by knockdown of AMPKa expression, indicating the involvement of AMPK signaling in this process. In line with increased IL-1 beta release, the ability of macrophages to kill engulfed bacteria was also intensified by berberine. This was corroborated by the in vivo finding that the peritoneal live bacterial load was decreased by berberine treatment. Moreover, berberine administration significantly improved survival of bacterial infected mice, concomitant with increased IL-1 beta levels and elevated neutrophil recruitment in the peritoneal cavity. Collectively, these data suggested that berberine could enhance bacterial killing by augmenting inflammasome activation in macrophages through AMPK signaling.