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Luteolin (Lut), a natural flavonoid extracted from plants, possesses several beneficial bioactivities. However, little is known about its anti-inflammatory properties. This study was designed to investigate the anti-inflammatory effects and underlying molecular mechanisms of luteolin in LPS plus IFN-γ activated RAW264.7 macrophages (M1 macrophages). RAW264.7 cells were induced to M1 polarization by LPS plus IFN-γ, accompanied with luteolin at non-cytotoxic doses. The results showed that the M1 macrophages presented dendrites characteristic with synapsis. In addition, luteolin can inhibit inflammation by modulating the expression of mediators, such as iNOS, IL-1β, IL-6, TNF-α, and CD86, whose expression was increased remarkably in the activated RAW264.7 cells. More importantly, the proteins p-STAT3-ser727 and p-STAT3-tyr705, which involved in inflammatory pathways, exhibited a dose-dependent degradation in M1 macrophages after treatment with luteolin. In conclusion, luteolin have potential applications as a functional food component in regulating inflammatory responses.