The physiological promotion effect of nanobubble (NB) water on living organisms is still a poorly understood phenomenon which was discovered 1 decade ago. Here, we analyzed the barley (Hordeum vulgare L.) embryo transcriptome following the exposure to NB water and low-concentration hydrogen peroxide (H2O2) using RNA-Seq. We found that 349 genes were differentially expressed after 24 h exposure to NB water and 97 genes were differentially expressed after exposure to H2O2 solution. Gene ontology enrichment and cluster analyses revealed that NB water induced expression of genes related to cell division and cell wall loosening. RNA-Seq, quantitative real-time polymerase chain reaction, and enzyme activity measurements all pointed to gene-encoding peroxidases as a major factor responsible for the effects of physiological enhancement due to NB water. The exogenous hydroxyl radical (center dot OH) produced by NB water significantly increased the expression of genes related to peroxidase and NADPH, thus leading to an increased endogenous superoxide anion (O-2(center dot-)) inside the barley seed. Appropriately, low concentrations of exogenously added reactive oxygen species (ROS) and endogenous ROS played important roles in plant growth and development. When ROS levels were low, the endogenous ROS was eliminated by ascorbate peroxidase and other peroxidases instead of activating the catalase and superoxidase dismutase. This data set will serve as the foundation for a system biology approach to understand physiological promotion effects of NB water on living organisms.