Plants take up nitrogen (N) both day and night. However, the molecular regulation of diurnal changes in N uptake is poorly understood. To test the hypothesis that light stimulates N uptake during the day, we examined the long-term effects of interrupted N supply (day only or night only) under hydroponic conditions. Seedlings experienced 32 days under daytime-fed (DF), nighttime-fed (NF), or continuous (CT) N supply. The root N uptake rate (NUR) differed between DF and NF from day 3 of treatment, after which NUR was significantly increased (by up to 82%) in DF and NF plants. The increased NUR during each half-day did not fully compensate for lost access to N during the other half-day, resulting in lower N accumulation by the end of the treatment. The reduction was smaller in DF plants, supporting the hypothesis that light promotes uptake. The root expression of ammonium transporter genes (OsAMT1;1, 1;2, 1;3) and a nitrogen-assimilation gene (OsGS1;2) was upregulated by 10% to 200% in both DF and NF plants relative to CT plants from day 15, more so in NF plants. No significant effect on OsG6PDH1 (in the pentose phosphate pathway) was detected.