5-Methyltetrahydrofolate (5-MTHF) is the major form of folate in human plasma and is the only folate form that can penetrate the blood-brain barrier. It has been widely used for the prevention and treatment of various diseases. It is mainly produced by chemical synthesis. However, the low production rate cannot meet the increasing demand. In addition, chemical synthesis is potentially detrimental to the environment. Despite various microorganisms synthetizing 5-MTHF, an efficient 5-MTHF bioproduction approach is lacking because of the tight regulation of the 5-MTHF pathway and limited metabolic flux toward the folic acid pathway. In this study, the 5-MTHF synthetic pathway in Bacillus subtilis was systematically engineered to realize 5-MTHF accumulation and further improve 5-MTHF production. Specifically, the 5-MTHF synthesis pathway with dihydrofolate (DHF) as the precursor was strengthened to shift the metabolic flux to 5-MTHF biosynthesis by replacing the native yitJ gene with Escherichia coli metF, knockout of purU, and overexpressing dfrA. The intracellular level of 5-MTHF increased 26.4-fold, reaching 271.64 mu g/L. Next, the 5-MTHF precursor supply pathway was strengthened by co-overexpression of folC, pabB, folE, and yciA. This resulted in a 93.2-fold improvement of the 5-MTHF titer, which reached 960.27 mu g/L. Finally, the clustered regularly interspaced short palindromic repeats interference system was used to identify key genes in the competitive and catabolic pathways for repression to further shift the metabolic flux toward 5-MTHF biosynthesis. The repression of genes thyA (existing in the purine metabolic pathway), pheA (existing in the competitive metabolic pathway), trpE (existing in the competitive metabolic pathway), and panB (existing in the pantoate synthesis pathway) significantly increased the titer of 5-MTHF. By repressing the pheA gene, the 5-MTHF titer reached 1.58 mg/L, which was 153.8-fold that of the wild-type strain of B. subtilis 168. Through medium optimization, the 5-MTHF titer reached 1.78 mg/L, which was currently the highest titer of 5-MTHF in B. subtilis. Apart from the highest titer of 5-MTHF, the highest titer of total folates including 5-MTHF, 5-FTHF, folic acid, and THF could reach 3.31 mg/L, which was 8.5-fold that in B. subtilis. To the best of our knowledge, the 5-MTHF and total folate titers reported here are the highest using a Generally regarded as safe (GRAS) bacterium as the production host. Overall, this study provides a good starting point for further metabolic engineering to achieve efficient biosynthesis of 5-MTHF by GRAS bacteria.