Abstract: Chicken embryo fibroblast DF-1 produces a large amount of lactate during cell culture in vitro. The accumulation of lactate not only accelerates the deterioration of culture environment, but also inhibits virus propagation. Therefore, improving the lactate metabolism and energy metabolism efficiency of DF-1 has some positive effects on viral vaccine production. In this study, mutation of Chicken Lactate Dehydrogenase (GgLDHA) gene in DF-1 based on CRISPR/Cas9 effectively reduced lactate accumulation, and significantly improved the energy metabolism of cells, and especially increased the ability of intracellular Infectious Bursal Disease Virus (IBDV) production. Firstly, with CBh promoter replaced by CMV, and human U6 (huU6) promoter exchanged into chicken U6 (chU6) promoter, the vector px458-chU6-CMV could be used for CRISPR/Cas9 gene editing suitable to DF-1. Compared to single sgRNA, the Insertion-deletion percentage (Indel%) was significantly increased from 5.4% to 26.2% by simultaneous co-transfection of two sgRNAs targeting GgLDHA gene. The genotype identification of 13 monoclonal cell lines with GgLDHA gene mutation showed that most gene mutations occurred near Double Strand Break (DSB) and led to deletion of the middle 29 bp fragment between two sgRNAs. Frameshift mutation was a main factor of targeted protein function inactivation. By mutating GgLDHA gene, the protein expression level and enzyme activity of GgLDHA were significantly reduced, compared to wild type. By studying the growth, glucose and lactate metabolism, energy metabolism and IBDV propagation in wild type and mutant clones, it was found that the GgLDHA mutation didn't affect cell growth, but lactate to glucose ratio was significantly reduced from 2.1 to 1.6, and the ATP synthesis rate was prominently increased by 40% (MC2) and 57% (MC5) compared with wild type cells respectively. Moreover, the increased aerobic metabolic rate flux and energy efficiency indicated that the energy metabolism efficiency of the mutant cell line was effectively increased. The IBDV titer of the mutant cell lines was significantly increased compared with wild type, and the highest IBDV titer could be up to about 5 times. In conclusion, GgLDHA mutation could effectively reduce the accumulation of lactate in DF-1 cell line, but ameliorate the state of energy metabolism, and effectively enhance the reproduction of IBDV virus, which provides a novel idea to improve the production capacity of vaccine against IBDV.