Berberine (BBR) is an effective drug for human intestinal inflammation by preventing intestinal adhesion of bacterial pathogens, while its antibacterial activity is ineffective. Although the antimicrobial mechanisms of BBR are intensively studied at high concentrations, the response of pathogens to its low concentrations remains poorly understood. Here we demonstrated that low concentrations of BBR (3 and 6 μg/mL) conferred by hormesis accelerated cell growth of an important Gram-negative pathogen, Klebsiella pneumoniae, in vitro, while higher concentrations (25 and 50 μg/mL) resulted in the opposite. Transcriptome analysis of K. pneumoniae revealed the up-regulated expression of the KmrA efflux pump and further confirmed it was hypersensitive to BBR stress. Strikingly, when cultivated in tetracycline, the growth-promoting effect of BBR became more significant, while this effect was reversed in the presence of the efflux pump inhibitor cyanide-m-chlorophenylhydrazone. The hormesis was also found in Enterobacter cloacae and Acinetobacter baumannii. More importantly, the presence of BBR at low concentrations resulted in higher minimal inhibitory concentrations of efflux-related antibiotics such as rifampicin and azithromycin. Overall, our data demonstrated the hormesis of BBR and revealed the potential risk of its applications against Gram-negative pathogens at low concentrations.