Luteolin, a plant flavonoid, has been shown to suppress inflammatory responses; however, the mechanism of luteolin on cardiac myocyte inflammation is still unknown. Because tumor necrosis factor-α (TNF-α), an inflammatory cytokine, is elevated in the failing heart and exerts multiple potentially harmful effects on cardiac myocytes, we therefore sought to examine the effects of luteolin on the expression of TNF-α in neonatal rat cardiac myocytes. In the present study, enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoblot, immunochemistry staining, and electrophoretic mobility shift assays (EMSA) were performed. ELISA assay showed that luteolin decreased lipopolysaccharide (LPS)-induced production of TNF-α in the medium. Real-time PCR assay confirmed that luteolin also inhibited LPS-induced increase in TNF-α mRNA in myocytes. Furthermore, immunoblot and immunochemistry staining assays represented that luteolin blocked LPS-induced IκB-β degradation and NF-κB p65 subunit nuclear translocation. In addition, EMSA demonstrated that luteolin reduced LPS-induced NF-κB DNA binding activity. Luteolin protects against LPS-induced TNF-α expression via inhibition of the NF-κB signaling pathway, suggesting that luteolin may be a potential therapeutic agent for the treatment of inflammation-related myocardial diseases.