The voltage-dependent anion channels (VDACs), prominently localized in the outer mitochondrial membrane, play important roles in the metabolite exchange, energy metabolism and mitochondria-mediated apoptosis process in mammalian cells. However, relatively little is known about the functions of VDACs in plants. To further investigate the function of AtVDAC1 in Arabidopsis, we analyzed a T-DNA insertion line for the AtVDAC1 gene. The knock-out mutant atvdac1 showed reduced seed set due to a large number of undeveloped ovules in siliques. Genetic analyses indicated that the mutation of AtVDAC1 affected female fertility and belonged to a sporophytic mutation. Abnormal ovules in the process of female gametogenesis were observed using a confocal laser scanning microscope. Interestingly, both mitochondrial transmembrane potential (ΔΨ) and ATP synthesis rate were obviously reduced in the mitochondria isolated from atvdac1 plants.