<jats:title>Abstract</jats:title> <jats:p>In this study, Corynebacterium glutamicum ATCC 13032 was engineered to produce l-citrulline through a metabolic engineering strategy. To prevent the flux away from l-citrulline and to increase the expression levels of genes involved in the citrulline biosynthesis pathway, the argininosuccinate synthase gene (argG) and the repressor gene (argR) were inactivated. The engineered C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2) produced higher amounts of l-citrulline (5.43 g/L) compared to the wildtype strain (0.15 g/L). To determine new strategies for further enhancement of l-citrulline production, the effect of l-citrulline on ornithine acetyltransferase (EC 2.3.1.35; OATase; ArgJ) was first investigated. Citrulline was determined to inhibit Ornithine acetyltransferase; for 50 % inhibition, citrulline concentration was 30 mM. The argJ gene from C. glutamicum ATCC 13032 was cloned, and the recombinant shuttle plasmid pXMJ19-argJ was constructed and expressed in C. glutamicum ATCC 13032 ∆argG ∆argR (CIT 2). Overexpression of the argJ gene exhibited increased OAT activity and resulted in a positive effect on citrulline production (8.51 g/L). These results indicate that OAT plays a vital role during l-citrulline production in C. glutamicum.