Because steroids and cyclooxygenase inhibitors may cause serious side effects, the IκB kinase (IKK) β/nuclear factor-κB (NF-κB) system has become an intriguing candidate anti-inflammatory target. Rhein, the active metabolite of diacerein, possesses anti-inflammatory ability with a gastrointestinal protective effect. However, in a preliminary study, we accidentally found that rhein showed both anti- and proinflammatory activities in lipopolysaccharide (LPS)-activated macrophages. Thus, in this study, we explored the underlying molecular mechanisms of the dual effects of rhein. In LPS-activated macrophages, rhein inhibits NF-κB activation and sequentially suppresses its downstream inducible nitric oxide synthase, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) transcription and supernatant nitric oxide and IL-6 levels by inhibiting IKKβ (IC50 ≈ 11.79μM). But in the meantime, rhein enhances the activity of caspase-1 by inhibiting intracellular (in situ) IKKβ, in turn increasing the IL-1β and high-mobility-group box 1 release, which can be amplified by rhein׳s reductive effect on intracellular superoxide anion. Unexpectedly, it is because of IKKβ inhibition that rhein significantly enhances TNF-α secretion and phagocytosis in macrophages with or without LPS. These results indicate that rhein exerts anti- and proinflammatory activities by targeting IKKβ inhibition, providing a molecular mechanism for the unanticipated role of rhein in macrophages. Furthermore, our study also highlights the potential complications of IKKβ inhibitor (e.g., rhein, diacerein, etc.) application in inflammation disorders, for the overall effects of IKKβ inhibition in various organ systems and disease processes are not easily predictable under all circumstances.