Cytidine is an important raw material for nucleic acid health food and genetic engineering research. In recent years, it has shown irreplaceable effects in anti-virus, anti-tumor, and AIDS drugs. Its biosynthetic pathway is complex and highly regulated. In this study, overexpression of uracil permease and a nucleoside transporter from Bacillus amyloliquefaciens related to cell membrane transport in Escherichia coli strain BG-08 was found to increase cytidine production in shake flask cultivation by 1.3-fold (0.91 +/- 0.03 g/L) and 1.8-fold (1.26 +/- 0.03 g/L) relative to that of the original strain (0.70 +/- 0.03 g/L), respectively. Co-overexpression of uracil permease and a nucleoside transporter further increased cytidine yield by 2.7-fold (1.59 +/- 0.05 g/L) compared with that of the original strain. These results indicate that the overexpressed uracil permease and nucleoside transporter can promote the accumulation of cytidine, and the two proteins play a synergistic role in the secretion of cytidine in Escherichia coli.