Nuclear factor (NF)-κB plays a pivotal role in the regulation of inflammatory response in macrophages. Berberine (BBR), which is an active constituent isolated fromCoptis rhizome, possesses a prominent anti-inflammatory activity. Here we show that BBR changes the global acetylation landscape in LPS-induced protein acetylation of macrophages and reduces the acetylation of NF-κB subunit p65 at site Lys310(p65Lys310), leading to the inhibition of NF-κB translocation and transcriptional activity to suppress the expressions of inflammatory factors. BBR resists the inflammatory response in acute LPS-stimulated mice through downregulation of p65Lys310acetylation in peritoneal macrophages. In obese mice, BBR alleviates the metabolic disorder and inflammation with the reduced acetylation of p65Lys310in white adipose tissue. Furthermore, we demonstrate that BBR acts as a regulator of p65Lys310by inhibiting the expression of p300 in macrophages. Our findings elucidate a new molecular mechanism for the anti-inflammatory effect of BBR via the p300/p65Lys310axis.