Macrophages are the main cells responsible for the innate immunity, and their activation by lipopolysaccharide (LPS) from Gram-negative bacteria or interferon (IFN)-γ from host immune cells is important for controlling infections. However, the overwhelming activation of macrophages can cause a severe inflammatory state. This study investigated the inhibitory mechanism of xanthohumol (XN) against the inflammatory effectors (IL-1β, TNF-α, and iNOS) in activated RAW264.7 macrophages by using different stimuli such as LPS, IFN-γ, or LPS plus IFN-γ. XN is a major prenylated chalcone found in hops, which is used to add bitterness and flavor to beer. XN reduced the expression of the LPS receptor components such as TLR4 and MD2 resulting in the suppression of NF-κB activation in LPS-activated RAW264.7 cells. In the IFN-γ stimulated RAW264.7 cells, the binding activity of STAT-1α and IRF-1 was inhibited by XN. This suggests that differential signaling pathways are used by XN for the inhibition of excess inflammatory mediators depending on the stimuli in macrophages.