Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathogen that causes severe community-acquired infections; however, the immune mechanisms controlling intracellular hvKp have yet to be clearly defined. In this study, we investigated the therapeutic potential of berberine against hvKp infection and elucidated the underlying molecular mechanisms using macrophage cell models and in vivo zebrafish models. Berberine significantly reduced intracellular hvKp survival in macrophages and improved survival in hvKp-infected zebrafish. Berberine markedly attenuated proinflammatory cytokine production and inhibited the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways. Notably, we found that hvKp exploited host lipid droplets (LD) biosynthesis to support its intracellular survival, and berberine effectively suppressed LD accumulation. Mechanistically, berberine promoted the nuclear translocation of transcription factor EB (TFEB), thereby enhancing lipolysis. Although berberine upregulated autophagy-related gene expression during hvKp infection, it did not induce lipophagy, the selective autophagic degradation of LD. Collectively, these findings indicate that berberine has potential as a therapeutic agent against hvKp infection by modulating host lipid metabolism to restrict bacterial intracellular survival.