The activity of alpha-1,2-mannosidase I is required for the conversion of high-mannose to hybrid-type (ConA reactive) and complex-type N-glycans (Phaseolus vulgaris-leukoagglutinin [PHA-L] reactive) during posttranslational protein N-glycosylation. We recently demonstrated that alpha-1,2-mannosidase I mRNA decreases in graft-infiltrating CD11c(+) dendritic cells (DCs) prior to allograft rejection. Although highly expressed in immature DCs, little is known about its role in DC functions. In this study, analysis of surface complex-type N-glycan expression by lectin staining revealed the existence of PHA-L-low and PHA-L-high subpopulations in murine splenic conventional DCs, as well as in bone marrow-derived DC (BMDCs), whereas plasmacytoid DCs are nearly exclusively PHA-L-high. Interestingly, all PHA-L-high DCs displayed a strongly reduced responsiveness to TNF-alpha-induced p38-MAPK activation compared with PHA-L-low DCs, indicating differences in PHA-L-binding capacities between DCs with different inflammatory properties. However, p38 phosphorylation levels were increased in BMDCs overexpressing alpha-1,2-mannosidase I mRNA. Moreover, hybrid-type, but not complex-type, N-glycans are required for TNF-alpha-induced p38-MAPK activation and subsequent phenotypic maturation of BMDCs (MHC-II, CD86, CCR7 upregulation). alpha-1,2-mannosidase I inhibitor-treated DCs displayed diminished transendothelial migration in response to CCL19, homing to regional lymph nodes, and priming of IFN-gamma-producing T cells in vivo. In contrast, the activity of alpha-1,2-mannosidase I is dispensable for LPS-induced signaling, as well as the DCs' general capability for phenotypic and functional maturation. Systemic application of an alpha-1,2-mannosidase I inhibitor was able to significantly prolong allograft survival in a murine high-responder corneal transplantation model, further highlighting the importance of N-glycan processing by alpha-1,2-mannosidase I for alloantigen presentation and T cell priming. The Journal of Immunology, 2011, 186: 5201-5211.