Artemisinin can be isolated from Artemisia annua L. In addition to its well-known anti-malarial activity, artemisinin has antitumor and anti-microbial effects. In this study, we investigated the effect of artemisinin on the production of IL-12p40, which is important in the generation of T helper 1 responses. Artemisinin significantly induced IL-12p40 production in LPS-stimulated RAW264.7 macrophage cells. To elucidate the signaling molecules regulated by artemisinin in induced IL-12p40 production, the DNA-binding activity of several transcription factors and activation of mitogen-activated protein kinase (MAPK)s were investigated. The band intensities of NF-κB, AP-1, and SP1, and the activation of p38 MAPK and ERK were not changed by artemisinin. However, the induced phosphorylation of JNK was significantly decreased by artemisinin, and inhibition of the JNK signaling pathway further increased IL-12p40 production in LPS-stimulated RAW264.7 macrophage cells. Taken together, these data suggest that artemisinin induces the production of IL-12p40 in LPS-stimulated macrophage cells by inhibiting JNK activity.