The role of 2-hydroxy-(4-methylseleno)butanoic acid (OH-SeMet), a form of organic selenium (Se), in selenoprotein synthesis and inflammatory response of THP1-derived macrophages stimulated with lipopolysaccharide (LPS) has been investigated. Glutathione peroxidase (GPX) activity, GPX1 gene expression, selenoprotein P (SELENOP) protein and gene expression, and reactive oxygen species (ROS) production were studied in Se-deprived conditions (6 and 24 h). Then, macrophages were supplemented with OH-SeMet for 72 h and GPX1 and SELENOP gene expression were determined. The protective effect of OH-SeMet against oxidative stress was studied in HO-stimulated macrophages, as well as the effect on GPX1 gene expression, oxidative stress, cytokine production (TNFα, IL-1β and IL-10), and phagocytic and killing capacities after LPS stimulation. Se deprivation induced a reduction in GPX activity, GPX1 gene expression, and SELENOP protein and gene expression at 24 h. OH-SeMet upregulated GPX1 and SELENOP gene expression and decreased ROS production after HO treatment. In LPS-stimulated macrophages, OH-SeMet upregulated GPX1 gene expression, enhanced phagocytic and killing capacities, and reduced ROS and cytokine production. Therefore, OH-SeMet supplementation supports selenoprotein expression and controls oxidative burst and cytokine production while enhancing phagocytic and killing capacities, modulating the inflammatory response, and avoiding the potentially toxic insult produced by highly activated macrophages.