<jats:title>Abstract</jats:title> <jats:p>L-citrulline plays an important role in human health and nutrition and is an intermediate of the L-arginine biosynthetic pathway. L-citrulline is a by-product of L-arginine production by <jats:italic>Corynebacterium glutamicum</jats:italic>. In this study, <jats:italic>C. glutamicum</jats:italic> was engineered for overproduction of L-citrulline as major product without L-arginine being produced as by-product. To this end, L-arginine biosynthesis was derepressed by deletion of the arginine repressor gene <jats:italic>argR</jats:italic> and conversion of L-citrulline towards L-arginine was avoided by deletion of the argininosuccinate synthetase gene <jats:italic>argG</jats:italic>. Moreover, to facilitate L-citrulline production the gene encoding a feedback resistant N-acetyl L-glutamate kinase <jats:italic>argB</jats:italic> <jats:sup>fbr</jats:sup> as well as the gene encoding L-ornithine carbamoylphosphate transferase <jats:italic>argF</jats:italic> were overexpressed. The resulting strain accumulated 44.1 ± 0.5 mM L-citrulline from glucose minimal medium with a yield of 0.38 ± 0.01 g⋅g<jats:sup>−1</jats:sup> and a volumetric productivity of 0.32 ± 0.01 g⋅l<jats:sup>−1</jats:sup>⋅h<jats:sup>−1</jats:sup>. In addition, production of L-citrulline from the alternative carbon sources starch, xylose, and glucosamine could be demonstrated.