The present study investigates As(V) toxicity in tomato (Solanum lycopersicum L. cv Pusa Rohini) and its alleviation by exogenous supplementation of nitrate. The seven days old seedlings were grown up to thirty days under defined levels of As(V) concentrations (0, 2.5, 6.25, and 12.5 mg/250 g soil) in alone or/and in combination with 20 mM nitrate. The arsenic accumulation, lipid peroxidation, DNA damage, photosynthesis, nitrogen assimilation, and AsA-GSH cycle were evaluated. Results revealed that As(V) exposure significantly (P ≤ 0.05) enhances the root, leaf and leaf sub-cellular arsenic accumulation, H2O2 and MDA contents in a dose-dependent manner. Comet assay indicated a progressive enhancement in the DNA damage with maximum tail length (58.33 ± 9.87 μm) and tail moment (25.05 ± 2.80) at 12.5 As(V) exposure. Nitrate supplementation counteracted As(V) toxicity on photosynthesis, nitrogen assimilation, and boosts AsA-GSH cycle at each respective As(V) treatments. The net photosynthesis was increased by 18% at 6.25 As(V), however, stomatal conductance and Fv/Fm were increased by 26%, and 11%, respectively, at 2.5 As(V) exposure. The activities of NR and GS were enhanced by 29% and 18%, respectively; contents of NO3-, NO2- and NH4+ were improved by 21%, 56%, and 13%, respectively, at 6.25 As(V) exposure. The activities of APX and GR were increased concomitantly with the ratios of AsA/DHA and GSH/GSSG. The study demonstrates that nitrate supplementation significantly (P ≤ 0.05) decreases As(V) accumulation, boosts the performance of AsA-GSH cycle, and consequently enhances the photosynthesis and nitrogen assimilation. Based on present findings, nitrate supplementation could be recommended as a promising approach to ameliorate the As(V) toxicity in plants.