Non-thermal plasma holds great potentials as an efficient, economical, and eco-friendly seed pretreatment method for improving the seed germination and seedling growth, but the mechanisms are still unclear. Therefore, a plant model organism Arabidopsis thaliana was used to investigate the physio-biochemical responses of seeds to non-thermal plasma at different treatment times by measuring the plant growth parameters, redox-related parameters, calcium (Ca2+) level and physicochemical modification of seed surface. The results showed that short-time plasma treatment (0.5, 1, and 3 min) promoted seed germination and seedling growth, whereas long-time plasma treatment (5 and 10 min) exhibited inhibitory effects. The level of superoxide anion (O-2(center dot-)) and nitric oxide (NO) and the intensity of infrared absorption of the hydroxyl group were significantly higher in short-time plasma treated Arabidopsis seeds, and the level of hydrogen peroxide (H2O2) was remarkably increased in long-time plasma treated seeds, indicating that O-2(center dot-), center dot OH, and NO induced by plasma may contribute to breaking seed dormancy and advancing seed germination in Arabidopsis, while plasma-induced H2O2 may inhibit the seed germination. The intensity of hydroxyl group and the contents of H2O2, malondialdehyde, and Ca2+ in Arabidopsis seedlings were obviously increased with the plasma treatment time. Catalase, superoxide dismutase, and peroxidase activities as well as proline level in short-time treated seedlings were apparently higher than in control. The etching effects of plasma on seed surface were dose-dependent, spanning from slight shrinkages to detached epidermis, which also significantly increased the oxidation degree of seed surface. Therefore, the improved activities of antioxidant systems, moderate center dot OH, H2O2, and Ca2+ accumulation and seed surface modification induced by plasma all contribute to the enhanced seedling growth of Arabidopsis after short-time plasma treatment.