Major histocompatibility complex class II (MHC II) is an antigen presenting molecule found on professional antigen presenting cells, such as dendritic cells (DCs). It plays a critical role in initiating adaptive immune responses by presenting antigen peptide to CD4+ T cells. The cell surface expression of MHC II is regulated by ubiquitination, with elevated MHC II expression observed in cells that lack MHC II ubiquitination. To investigate the impact of this ubiquitination on immune outcomes, we have characterised DCs in mice lacking MHC II ubiquitination (MHCIIKRKI/KI mice). Immunisation of MHCIIKRKI/KI mice with soluble, cell-associated and receptor-targeted ovalbumin has revealed a critical role for MHC II ubiquitination in T cell priming, with significant defects in MHC II presentation observed. A similar, albeit less significant, trend was observed for MHC I presentation. The decrease in T cell priming was accompanied by a decrease in splenic conventional DC (cDC) numbers. To investigate if the defect in T cell priming was due to changes in DC numbers in vivo, ex vivo antigen presentation assays were carried out with equal number of sorted DCs and T cells. This too showed significant defects in MHC II (and MHC I) presentation indicating an intrinsic defect in antigen presentation. In an effort to understand how the lack of MHC II ubiquitination impairs DC function, the biology of MHCIIKRKI/KI DCs was further examined. MHCIIKRKI/KI cDC subset 1 (cDC1) secrete significantly increased pro-inflammatory cytokines than wild type cDC1. In addition, MHCIIKRKI/KI DC have reduced proteolysis of DQ-ovalbumin. Interestingly, this was not due to decreased antigen uptake (as determined by the uptake of ovalbumin-Cy5). Overall, our data suggests an important role for MHC II ubiquitination in regulating DC function and immune outcomes.