ABSTRACT The adverse effects of intake nitrite upon human health are well known. However, eating fruits and vegetables is one of the main pathways to absorb nitrite because of nitrogen assimilation in plants. This study demonstrated that during storage of tomato fruit, the production of endogenous hydrogen (H2) was decreased, in parallel with nitrite accumulation and the senescence rate. Furthermore, exogenously applied H2 could delay the decreased fruit H2 production and senescence, but importantly, nitrite accumulation was blocked. Consistently, the activities and transcripts of nitrate reductase (NR; catalyzing the synthesis of nitrite) and nitrite reductase (NiR; responsible for the reduction of nitrite to ammonium), were either inhibited or increased, respectively, by 0.585 mM H2. Decreased or increased nitrite synthesis was observed when tungstate (an inhibitor of NR) or 2,6-dichloroindophenol sodium salt (a putative inhibitor of H2 synthesis) were applied separately. Time-course analysis revealed that the decrease in vitamin C, a well-known nitrite scavenger, was blocked by H2. Overall, this study strongly revealed that nitrite accumulation during storage of tomato fruit was prevented by H2. This study describes potential applications for H2 in agriculture and food industry, especially in the preservation of fruit and vegetable products. Abbreviations: Ar, argon; DCPIP, 2,6-dichloroindophenol sodium salt; GC, gas chromatograph; H2, hydrogen gas; HPLC, high-performance liquid chromatography; HRW, hydrogen-rich water; N, Newton; N2, nitrogen; NO, nitric oxide; NiR, nitrite reductase; NO2 −, sodium nitrite; NO3 −, sodium nitrate; NR, nitrate reductase; ONOO−, peroxynitrite anion; qPCR, Real-time quantitative reverse transcription-PCR; RNS, reactive nitrogen species; ROS, reactive oxygen species.