Ascorbate and tocopherol are important hydrophilic or lipophilic antioxidants in plants, while their crucial roles in the antioxidant defense system under ultraviolet B radiation were not well understood. The mutants of Arabidopsis thaliana deficient in ascorbate (vtc1 and vtc2) or tocopherol (vte1) were used to analyze their physiological, biochemical and metabolic change in responses to Ultraviolet B radiation. Results showed that loss of either ascorbate or tocopherol caused reduction in phenylpropanoid and flavonol glycosides compounds, as well as reduction in superoxide dismutase activity and total cellular antioxidant capacity. This ultimately led to higher oxidative stress as well as lower levels of photosynthetic pigments (carotenoid and chlorophyll) and CO2 assimilation rate in the vtc1, vtc2, and vte1 mutants than the wild type under UV-B radiation, besides unstable early light-induced protein (ELIP1) in those mutants. On the other hand, the loss of tocopherol in vte1 mutants was compensated by the increase of zeaxanthin and anthocyanin contents, which armed vte1 mutants with higher heat dissipation capacity in PS II and higher antioxidative capacity than vtc mutants. Consequently the tolerance to UV-B radiation were much higher in vte1 mutant than in vtc mutants, furthermore, PS II function and light harvesting protein (LHCb1) abundance were reduced only in ascorbate-deficient mutant relative to wild type. Our results suggested that the ascorbate and tocopherol provided not only direct protective function against UV-B radiation but also indirect effects by influencing other protective system, in particular by affecting the stability of various carotenoid and phenylpropanoid compounds.