To establish the cells stably co-expressing NOD1 receptor and enhanced green fluorescent protein (EGFP)-tagged nuclear factor of activated T cells 2 nuclear factor (NFAT2) (EGFP-NFAT2) in U2OS cell, the NOD1 (NM_006092) pcDNA3.1-3×Flag-hygro recombinant plasmid was transfected into U2OS-EGFP-NFAT2 cells, which were screened by pressure of hygromycin B and then incubated with NOD1 agonist lipopolysaccharides (LPS) for 30 min, and the green fluorescence intensity in the nucleus of the cells was detected by the high content screening assay. There were 46 cell strains expressing NOD1 in U2OS-EGFP-NFAT2 cells by EGFP-NFAT2 nuclear translocation assay. Among these cells, cells No 4 had the highest nuclear translocation function. Therefore, it was selected as the U2OS-EGFP-NFAT2-NOD1 cell for functional validation. The expression levels of NOD1 mRNA and protein in the selected U2OS-EGFP-NFAT2-NOD1 cells and the control cell U2OS-EGFP-NFAT2 were examined by real-time quantitative PCR (RT-qPCR) and Western blot. The results showed that NOD1 mRNA was stably expressed in this stably transfected cell line for 5-20 generations, and NOD1 protein was expressed in U2OS-EGFP-NFAT2-NOD1 stably transfected cell line, whereas no NOD1 protein was expressed in the control cell U2OS-EGFP-NFAT2. U2OS-EGFP-NFAT2-NOD1 cells were treated with histones or LPS for 30 min, and the EGFP-NFAT2 nuclear translocation was detected by the high content screening assay. Histones were found to significantly increase the EGFP-NFAT2 nuclear translocation in U2OS-EGFP-NFAT2-NOD1 stably transfected cells over a range of concentrations. The U2OS-EGFP-NFAT2-NOD1 cells were divided into the solvent control group, NOD1 receptor antagonist nodinitib-1+histone group, and histone group. The drug incubation time was 30 min, and the specificity of the NOD1 cells was verified by observing the EGFP-NFAT2 nuclear translocation through the high content screening assay. Compared with the histones group, the nodinitib-1+histones group significantly decreased EGFP-NFAT2 nuclear translocation in U2OSEGFP-NFAT2-NOD1 cells (P < 0.05). In conclusion, U2OS-EGFP-NFAT2-NOD1 cells stably co-expressing NOD1 and EGFP-NFAT2 are established, which can be used for screening antagonistic compounds targeting NOD1 pathogenic microorganisms with mechanism study.