In a previous work, an Escherichia coli strain with enhanced arginine production ability was constructed. In this study, we sought to optimize the fermentation process with respect to nitrogen supply. Nitrogen is an essential nutrient for cell growth. Furthermore, ample supply of nitrogen is particularly critical for arginine production as arginine is the most nitrogen-rich amino acid with 4 atoms of nitrogen per molecule. Besides, optimization of the nitrogen supply is also crucial as remaining nitrogen in the broth at the end of the fermentation makes the purification of arginine more complex. The effect of five inorganic nitrogen sources was studied: ammonium sulfate, ammonium phosphate, ammonium chloride, ammonium nitrate and ammonium hydroxide. Different carbon to nitrogen (C:N) ratios, ranging from 2.5 to 10, were investigated, with glucose as carbon source. Too high concentrations of nitrogen might have a detrimental effect on cell growth. Therefore, the same C:N ratios were assessed at both high (30 g/L) and low (15 g/L) glucose concentration. Fermentations were performed in 1-L bioreactors, using a minimal medium to avoid any other nitrogen source in the fermentation broth.