To construct a Bacillus subtilis strain for improved uridine production. The AAG(2846-2848) fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation. This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.