Nitrogen greatly impacts plant growth and development. The objective of this study was to characterize growth, nitrogen use, and gene expression of perennial ryegrass (Lolium perenne) in response to increasing nitrogen supplies. Perennial ryegrass (cv. Inspire) was grown in sand culture and irrigated with a half-Hoagland solution amended with 0, 0.5, 1.0, 2.5, 5.0, and 7.5 mm nitrogen. Leaf tissues were harvested at 10 days (first cutting) and 20 days (second cutting) and roots were harvested at 20 days. The relatively higher N supply (2.0–7.5 mm) resulted in a larger amount of leaf fresh and dry weight but lower root fresh and dry weight, especially for the second cutting. Root:leaf ratio was higher under low N, but lower under the high N treatment. Leaf N content was relatively higher under 2.5, 5, and 7.5 mm N than under the other three treatments, while 2.5 mm N exhibited relatively higher leaf carbon content for both cuttings. Leaf C:N ratio and leaf nitrogen use efficiency (LNUE) decreased with increasing N supplies for the first cutting but were higher under low N (0–1.0 mm) for both cuttings. Leaf C:N ratio and LNUE did not differ among low N and LNUE also remained unchanged among high N for the second cutting. Root N content increased, but the root C:N ratio and root N use efficiency (RNUE) decreased with increasing N supplies, especially under 2.5, 5.0, and 7.5 mm N. Low (0.5 mm), moderate (2.5 mm), and high (7.5 mm) N were chosen to examine the expression level of NR encoding nitrate reductase and GS1b encoding glutamine synthetase. Treatment of 0.5 mm N had higher expression levels of leaf NR than other two treatments for both cuttings and a higher level of leaf GS for the second cutting. Expression of NR in the roots did not vary among treatments but the expression of GS increased under 2.5 and 7.5 mm, compared with the 0.5 mm N. Differential leaf and root growth and physiological responses to low N (0 to 1 mm) and to moderate to high N (2.5 to 7.5 mm) could be used for examining the natural variation of N use in diverse perennial ryegrass populations.