Nitrogen (N) is one of the essential macronutrients that plays important roles in plant growth and development. To better understand the response of antioxidant system and N metabolism under N starvation and re-supply condition, physiological and transcriptomic analysis were performed in tomato roots. The malondialdehyde (MDA) and reactive oxygen species (ROS) contents increased significantly in tomato seedlings after N starvation for 24 h. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR), the ratio of ASA/DHA and GSH/GSSG, the NO3- contents, nitrate reductase (NR) activity were decreased after N starvation treatment and increased after N re-supply for 24 h. Compared with the control, 1766 genes were up-regulated and 2244 genes were down-regulated after N starvation in tomato. These differentially expressed genes (DEGs) are mainly enriched in functional items such as cellular process, metabolic process and catalytic activity. The KEGG pathways revealed that the DEGs were mainly involved in phenpropane biosynthesis, amino sugar and nucleotide sugar metabolism, and N metabolism. The expression patterns of tomato SlSOD, SlCAT, SlAPX, SlMDHAR, thioredoxin (SlTrxh), peroxiredoxin (SlPrx) and glutaredoxin (SlGrx) genes, and nitrate transporter SlNRT2.4, SlNR, glutamine synthetase (SlGS2), nitrite reductase (SlNiR) decreased after N starvation and increased after N re-supply, which were validated by qRT-PCR. Our results provide a basis for understanding the response of tomato to N deficiency and re-supply and a theoretical reference for cultivation regulation.