Abstract Mulberry leaf has been traditionally used to treat chronic diseases such as diabetes and cancer. The effect and mechanism of mulberry leaf extract (MLE) in LPS-induced activation of macrophage was investigated by the levels of production of proinflammatory mediators and cytokines, and their transcriptions. MLE effectively decreased proinflammatory mediators and cytokines production. Their expression levels were decreased at both transcript and protein levels by the pretreatment of MLE. Since all these genes are transcribed by NF-κB and MAPK, their activities were also examined. NF-κB activity was proved to be reduced by the preincubation with MLE through inhibition of IκBα phosphorylation. However, MLE-treatment had no effect on phosphorylation levels of MAPK. There results suggest that MLE can be used as an anti-inflammatory agent to inhibit NF-κB-mediated inflammatory response. In addition, our data support the idea that MLE can inhibit the activities of proinflammatory mediators and cytokines to ameliorate the disease conditions.