Background: Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammation that is a risk factor formany gastrointestinal cancers. Exosomes are gradually gaining attention as an emerging treatment method for IBD due to their important biological characteristics. NF-κB is an important pro-inflammatory transcription factor kept inactive by IκB protein in the cytoplasm by masking the nuclear localization signal of NF-κB. The deterioration of IκB ismainly ubiquitination, and this depends on neddylation. Methods: In this study, we established a dextran sulfate sodium (DSS)-induced IBD model in BABL/C mice to evaluate the effect of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-exosomes, hucMSC-Ex) on the repair of IBD.At the same time, human colorectalmucosa cells (FHC)were stimulated by LPS (lipopolysaccharide) in vitro to activate the inflammatory environment to study the mechanism of hucMSC-Ex regulating neddylation. The microRNA (miRNA) obtained by sequencing and transfection with hucMSC-Ex was used to verify the role of miR-326/neddylation/IκB/NF-κB signaling pathway in IBD repair. Results: HucMSC-Ex inhibited the process of neddylation in relieving DSSinduced IBD in mice. The binding of NEDD8 (neural precursor cell-expressed, developmentally downregulated gene 8) to cullin 1 and the activation of NF-κB signaling pathway were suppressed along with reduced expression levels of neddylation-related enzyme molecules. The same phenomenon was observed in FHC cells. The miRNA comparison results showed that miR-326 was highly expressed in hucMSC-Ex and played an important role in inhibiting the neddylation process. The therapeutic effect of hucMSC-Ex with high expression of miR-326 on IBD mice was significantly stronger than that of ordinary hucMSCEx. Conclusions: HucMSC-Ex relieves DSS-induced IBD in a mouse model by inhibiting neddylation through miR-326.