<jats:title>Abstract</jats:title> <jats:p>There are four nitrogen atoms in l-arginine molecule and the nitrogen content is 32.1%. By now, metabolic engineering for l-arginine production strain improvement was focused on carbon flux optimization. In previous work, we obtained an l-arginine-producing Corynebacterium crenatum SDNN403 (ARG) through screening and mutation breeding. In this paper, a strain engineering strategy focusing on nitrogen supply and ammonium assimilation for l-arginine production was performed. Firstly, the effects of nitrogen atom donor (l-glutamate, l-glutamine and l-aspartate) addition on l-arginine production of ARG were studied, and the addition of l-glutamine and l-aspartate was beneficial for l-arginine production. Then, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in ARG for increasing the l-glutamine and l-aspartate synthesis, and the l-arginine production was effectively increased. In addition, the l-glutamate supply re-emerged as a limiting factor for l-arginine biosynthesis. Finally, the glutamate dehydrogenase gene gdh was co-overexpressed for further enhancement of l-arginine production. The final strain could produce 53.2 g l−1 of l-arginine, which was increased by 41.5% compared to ARG in fed-batch fermentation.