Abstract: In order to construct a food-safe strain that could produce glycolate, the metabolic modification of Bacillus subtilis was carried out. In this study, the exogenous isocitrate lyase gene (aceA) was first integrated into the genome of Bacillus subtilis by homologous recombination, and the starting strain 164MCT-GA was constructed. Then the glycolate anabolism was optimized by means of metabolic engineering in the starting strain 164MCT-GA. The results showed that 164MCT-GA could synthesize glycolate with glycerol as substrate, and the yield of shaker fermentation was 0.114 g/L. To increase the supply of the key intermediate substrates, the citrate synthase gene (citA) and the glyoxylate reductase gene (yvcT) were overexpressed by replacing the native promoter with individual T7 promoter. The Bacillus strains were further engineered at multiple loci that included lactate dehydrogenase (ldh), phosphate acetyltransferase (pta) and acetyl-CoA transacetylase (mmgA, yhfs), in an attempt to modulate the carbon flux toward the formation of glycolate with a higher efficiency. The fermentation study revealed that the accumulated concentration of glycolate from the obtained B. subtilis strain GA3-52 reached 0.572 g/L, with a conversion rate of 0.175 g/g glycerol, the titer was more than five times as much as that achieved by 164MCT-GA. Thus, this study constructed a de novo synthesis pathway in B. subtilis, and laid the foundation for the fermentation production of high yield glycolic acid by food safety bacteria.