Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation.