Objectives The metabolic pathway related to uridine production was modified in Bacillus subtilis in order to increase the production of uridine. Results Decreasing the relative transcriptional level of pur operon in Bacillus subtilis TD300 to 80%, and the production of the derived strain TD312 was increased to 11.81 g uridine/l and the yield was increased to 270 mg uridine/g glucose. The expression of pucR gene in situ by P-ccpA resulting in a 194.01-fold increase in the relative transcriptional level of pucR gene and 349.71-fold increase in the relative transcriptional level of ure operon, respectively. Furthermore, the production of TD314 reached 13.06 g uridine/l, while the yield reached 250 mg uridine/g glucose. Conclusion This is the first report that more than 13 g uridine/l with a yield of 250 mg uridine/g glucose is produced in shake flask fermentation of genetically engineered Bacillus subtilis.