Arabidopsis thaliana had been selected as the research material in this study, to test the darkness stress on the eco-physiological characteristics (including phenotype, total ascorbate content and the chlorophyll fluorescence) of three genotype plants with different endogenous ascorbate contents (GLDH gene over-expression named as gldh236OE, wide type Col and the AsA deficient mutants vtc2-1). The results indicated that the contents of total ascorbic acid of the three genotype plants were trended down under the darkness. Compared with the wide type (Col), total ascorbate content of vtc2-1 was significantly reduced (P005). The leaf etiolation speed of vtc2-1 was faster than Col, but gldh236OE was slower. The darkness induced the decrease of Fv/Fm and Yield, which hindered photosynthetic efficiency of PSII. It also reduced the NPQ of vtc2-1, and exogenous applied ascorbic acid markedly enhanced its capability of the heat dissipation (P005). The present results indicated that the deficient of endogenous ascorbate reduced plants resistance to dark stress and exogenous ascorbate relieved the stress of the dark in part.