Physiological selenium (Se) levels counteract excessive inflammation, with selenoproteins shaping the immunoregulatory cytokine and lipid mediator profile. How exactly differentiation of monocytes into macrophages influences the expression of the selenoproteome in concert with the Se supply remains obscure. THP-1 monocytes were differentiated with phorbol 12-myristate 13-acetate (PMA) into macrophages and (i) the expression of selenoproteins, (ii) differentiation markers, (iii) the activity of NF-κB and NRF2, as well as (iv) lipid mediator profiles were analyzed. Se and differentiation affected the expression of selenoproteins in a heterogeneous manner. GPX4 expression was substantially decreased during differentiation, whereas GPX1 was not affected. Moreover, Se increased the expression of selenoproteins H and F, which was further enhanced by differentiation for selenoprotein F and diminished for selenoprotein H. Notably, LPS-induced expression of NF-κB target genes was facilitated by Se, as was the release of COX- and LOX-derived lipid mediators and substrates required for lipid mediator biosynthesis. This included TXB2, TXB3, 15-HETE, and 12-HEPE, as well as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Our results indicate that Se enables macrophages to accurately adjust redox-dependent signaling and thereby modulate downstream lipid mediator profiles.