Objective To investigate the relationship between the expression of nuclear factor ⅠB (NFIB) in myeloid cells and intestinal inflammation by constructing NFIB conditional gene knockout (cKO) mice. Methods Human Protein Atlas database, Genotype-Tissue Expression database, and FANTOM5 database were used to investigate the expression of NFIB in inflammatory cells. NFIB-floxed mice were constructed using CRISPR/Cas9 technology and hybridized with LyZ2-Cre transgenic mice. Myeloid specific NFIB cKO mice (NFIB^fl/flLyz2-Cre) were obtained by self-crossing the progeny. After the genotype identification of mice by agarose gel electrophoresis, 4 NFIB cKO mice of C57BL/6N strain were selected as experimental group, and 4 non-cKO mice were selected as control group. Both groups were induced with 2.5% dextran sulfate sodium salt (DSS) under the same condition to establish a chronic colitis model, and the severity of colitis was evaluated by clinical manifestations and histopathology. Results Analysis showed that NFIB was expressed in both myeloid granulocytes and monocytes, and the highest expression was found in neutrophils. NFIB cKO mice were successfully constructed using CRISPR/Cas9 technology and Cre-loxP system. DSS-induced enteritis NFIB cKO mice developed diarrhea, gross blood stools, reduced activity, and weight loss in a short time. The gross examination of the intestines showed that the colon of the NFIB cKO mice was significantly shorter than that of the non-cKO mice (［8.23±0.35］ cm vs ［10.30±0.36］ cm, P＜0.01). Intestinal H-E staining showed changes in mucosal glandular structure and connective tissue hyperplasia with extensive inflammatory cell infiltration in NFIB cKO mice. The histological score of NFIB cKO mice was significantly higher than that of non-cKO mice (4.25±0.50 vs 0.50±0.58, P＜0.01). Intestinal immunohistochemical staining showed that more CD11b positive cells were recruited in NFIB cKO mice than non-cKO mice. Conclusion Myeloid specific NFIB cKO mice have been successfully constructed, and NFIB in myeloid cells can reduce infiltration of immune cells (granulocytes or/and monocytes) to inhibit intestinal inflammation.