Thymidine is an important precursor in antiviral drugs. We have enhanced thymidine production in E. coli by eliminating the repressors in the transcription of the gene coding for carbamoyl phosphate synthetase. The operon for carbamoyl phosphate synthetase (CarAB) in the thymidine biosynthesis regulatory pathway was derepressed by disrupting three known repressors (purR, pepA and argR). Combinatorial disruption of three repressors increased CarA expression levels in accordance with degree of disruption, which had a positive correlation with thymidine production. By simultaneous disruption of three repressors (BLdtugRPA), CarA expression level was increased by 3-fold compared to the parental strain, leading to an increased thymidine yield from 0.25 to 1.1 g thymidine l(-1). From BLdtugRPA, we established BLdtugRPA24 by transforming two plasmids expressing enzymes in the thymidine biosynthetic pathway and obtained 5.2 g thymidine l(-1) by Ph-stat fed-batch fermentation.