Eating fruits and vegetables loaded with natural antioxidants can boost human health considerably and help fight off diseases linked to oxidative stress. Hydrogen has unique antioxidant effects. However, its low-solubility and fast-diffusion has limited its applications in agriculture. Integration of hydrogen with nanobubble technology could address such problems. However, the physiological adaptation and response mechanism of crops to hydrogen nanobubbles is still poorly understood. Antioxidant concentrations of lycopene, ascorbic acid, flavonoids, and resveratrol in hydrogen nanobubble water drip-irrigated tomato fruits increased by 16.3–264.8% and 2.2–19.8%, respectively, compared to underground water and oxygen nanobubble water. Transcriptomic and metabolomic analyses were combined to investigate the regulatory mechanisms that differed from the controls. Comprehensive multi-omics analysis revealed differences in the abundances of genes responsible for hormonal control, hydrogenase genes, and necessary synthetic metabolites of antioxidants, which helped to clarify the observed improvements in antioxidants. This is the first case of hydrogen nanobubble water irrigation increasing numerous natural antioxidant parts in fruits. Considering the characteristics of hydrogen and the application of the nanobubble technology in agriculture, the findings of the present study could facilitate the understanding of the potential effects of hydrogen on biological processes and the mechanisms of action on plant growth and development.

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