In previous studies, selenium (Se) was reported to play critical roles in anti-inflammatory activities. Nevertheless, limited information could be obtained during inflammation about selenomethionine (SeMet) in U937 human macrophage cells. The purpose of this study was to investigate the effects of SeMet on the inflammatory responses to lipopolysaccharide (LPS)-induced U937 macrophage cells and the signaling pathways targeted. U937 cells were pretreated with SeMet (1 μM) and subsequently induced with LPS (1 μg/ml) for 24 h. In the cell counting kit-8 assay (CCK-8), SeMet significantly inhibits the proliferation of U937 cells. SeMet also inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) stimulated by LPS. In the Western blot assay and real-time polymerase chain reaction (RT-PCR), SeMet significantly reduced protein expression and production of inducible NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), and COX-2 in U937 cells. Furthermore, SeMet markedly suppressed the LPS-mediated activation of nuclear factor-kappa B (NF-κB) by blocking the degradation of inhibitor-κB proteins (IκBα) and lessening the translocations of P50 subunit content of NF-κB in the nucleus. These findings suggested the anti-inflammatory activity of SeMet in U937 cells; indicating that SeMet might be a potential treatment for inflammation therapy.