Abstract: This study was conducted to explore the effect of interferon gene stimulating factor (STING) gene knockout on replication of pseudorabies virus (PRV). The STING gene of porcine pulmonary macrophage cell line 3D4/21 was knocked out by using CRISPR/Cas9 technique, the target gene knockout efficiency was detected by using T7 nuclease, and the activity of gene knockout cells was detected by using cell counting kit. The recombinant virus PRV-GFP expressing green fluorescent protein (GFP) was used to infect the knockout cells, and the fluorescence intensity of the infected cells was detected by flow cytometry. The expression of gB and TK genes in PRV and the transcription of IL-1β, IFN-β and ISG15 genes in infected cells were detected by quantitative RT-PCR, the expression level of PRV gE protein was detected by Western blot, and the viral titer of offspring was determined by viral titration. The results showed that all of three designed exon2 specific sgRNA of STING gene could cleave the target sequences of 3D4/21 cells, and the gene editing efficiency of sgRNA3 was the highest. SgRNA1-mediated STING gene knockout lines were cloned and three gene knockout cell lines were obtained. Gene knockout had no effect on cell activity. PRV-GFP positive cells cultured in parental cells accounted for 80.77%, and PRV-GFP positive cells cultured in STING knockout cells accounted for 95.55%. STING gene knockout could promote the expression of PRV gene. The virus titer of parent 3D4/21 cells was 10^6.2 TCID₅₀·0.1 mL^-1, and the virus titer of STING knockout cells was 10^8.3 TCID₅₀·0.1 mL^-1. The transcription of IL-1β, IFN-β and ISG15 in virus infected cells were significantly down-regulated. STING gene knockout can promote the replication of PRV in 3D4/21 cells, which may be related to the inhibition of IL-1β, IFN-β and ISG15 expression in infected cells.