Post-translational protein modifications (PTM) are necessary for the proper function of cells. The role of PTMs in regulating immune responses, specifically those mediated by dendritic cells (DCs), which are critical for both innate and adaptive immunity, is not well understood. Utilizing multiple, but complementary approaches, we determined the role of an important but less understood type of PTM, namely neddylation, in regulating DC functions. Inhibition of neddylation suppressed the release of proinflammatory cytokines by DCs in response to TLR, NLR and non-infectious CD40L stimulation. These effects were more profound than those mediated by the proteasome inhibitor bortezomib or a commonly used anti-inflammatory agent, dexamethasone. Targeting neddylation also suppressed the ability of DCs to stimulate murine allogeneic T cells in vitro and in vivo and human allogeneic T cell responses in vitro. Mechanistic studies demonstrated that inhibition of neddylation reduced both canonical and non-canonical NF-κB activity. Neddylation inhibition prevented the degradation of IκB and thus reduced the translocation and activation of NF-κB, but without perturbation of the MAPK/ERK pathway. Thus blocking neddylation could be a novel strategy for mitigating immune mediated disease processes.