Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine‐inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL‐1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre‐clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7‐specific inhibitor, has limited effects on IL‐6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co‐inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU‐001i or ML604440 impairs MHC class I cell surface expression, IL‐6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co‐inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases. Synopsis Simultaneous targeting of the immunoproteasome subunits LMP2 and LMP7 is required for optimal therapeutic efficacy in the treatment of autoimmunity. These findings will promote the design of novel immunoproteasome inhibitors with optimal therapeutic efficacy. Simultaneously targeting the immunoproteasome subunits LMP2 and LMP7 blocks autoimmunity. Co‐inhibition of LMP2 and LMP7 is required to reduce MHC‐I surface expression, IL‐ 6 production, and Th17 differentiation. Co‐inhibition strongly ameliorates disease phenotypes of experimental colitis and EAE. Simultaneous targeting of the immunoproteasome subunits LMP2 and LMP7 is required for optimal therapeutic efficacy in the treatment of autoimmunity. These findings will promote the design of novel immunoproteasome inhibitors with optimal therapeutic efficacy.