Berberine (BBR) has been shown to improve several metabolic disorders such as obesity, type 2 diabetes, and dyslipidemia by stimulating AMP-activated protein kinase (AMPK). However, the effects of BBR on pro-inflammatory responses in macrophages are poorly understood. Here we show that BBR represses pro-inflammatory responses through AMPK activation in macrophages. In adipose tissue of obese db/db mice, BBR treatment significantly down-regulated the expression of pro-inflammatory genes such as TNF, IL-1, IL-6, MCP-1, iNOS and COX2. Consistently, BBR inhibited LPS-induced expression of pro-inflammatory genes including IL-1, IL-6, iNOS, MCP-1, COX 2, and MMP9 in peritoneal macrophages and RAW 264.7 cells. Upon various pro-inflammatory signals including LPS, free fatty acids, and hydrogen peroxide, BBR suppressed the phosphorylation of MAPKs such as p38, ERK, and JNK, and the level of reactive oxygen species in macrophages. Moreover, these inhibitory effects of BBR on pro-inflammatory responses were abolished by AMPK inhibition via either Compound C, an AMPK inhibitor, or dominant-negative AMPK, implying that BBR would down-regulate pro-inflammatory responses in macrophages via AMPK stimulation.