Deubiquitination of NF-κB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The role of the naturally occurring CYLD splice variant in dendritic cell (DC) function was analyzed using CYLDex7/8 mice, which lack the full-length CYLD (flCYLD) transcript and overexpress the short splice variant (sCYLD). Bone marrow–derived DCs from CYLDex7/8 mice display a hyperactive phenotype in vitro and in vivo and have a defect in establishing tolerance with the use of DEC-205–mediated antigen targeting to resting DCs. The combination of sCYLD overexpression and lack of flCYLD in CYLDex7/8 DCs leads to enhanced NF-κB activity accompanied by an increased nuclear translocation of the IκB molecule Bcl-3, along with nuclear p50 and p65. This suggests that, in contrast to flCYLD, sCYLD is a positive regulator of NF-κB activity, and its overexpression induces a hyperactive phenotype in DCs.