Graft-versus-host disease (GVHD) remains one of the major complications after allogeneic hematopoietic stem cell transplantation that is mainly treated with glucocorticoids such as prednisolone. In this study the influence of monocyte subpopulations, prednisolone, and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)₂D3) on the induction of a proinflammatory subset of Th17 cells (MDR⁺Th17.1) characterized by CCR6⁺CXCR3^hiCCR4^loCCR10⁻CD161⁺ and stable expression of the multidrug resistance protein type 1 (MDR1) was investigated. Our results demonstrate that intermediate monocytes are increased in patients with acute GVHD, promoting the induction of proinflammatory MDR1⁺Th17.1 cells. Furthermore, prednisolone induces the development of MDR1⁺Th17.1 cells, whereas 1α,25-(OH)₂D3 acts as an anti-inflammatory, leading to diminished percentages of proinflammatory MDR1⁺Th17.1 cells in the presence of prednisolone after stimulation with the TLR4-ligand S100A8/S100A9. Moreover, 1α,25-(OH)₂D3 decreased the expression level of the targets JAK2 and CD74, both associated with T cell activation, in monocytes. Thus, in steroid-resistant GVHD, 1α,25-(OH)₂D3 could be an important regulator in monocyte-induced development of proinflammatory MDR1⁺Th17.1 cells and might therefore be a potential therapeutic agent in combination with glucocorticoids for GVHD treatment.