In flowering plants, pollen formation is a complex process and strictly controlled by genetic factors. Although thousands of genes have been identified to be highly or specifically expressed in pollen grains, little is known about the functions and regulatory mechanisms of the genes in pollen formation. Here we report the characterization of a novel gene, MALE GAMETOPHYTE DEFECTIVE 1 (MGP1), that is essential for pollen formation in Arabidopsis thaliana. MGP1 encodes the F(A)d subunit of mitochondrial F(1)F(0)-ATP synthase in Arabidopsis. It was highly expressed in pollen grains at the later developmental stage. Mutation in MGP1 led to destruction of the mitochondria in pollen grains at the dehydration stage and subsequently death of the pollen grains. These results suggested that MGP1 plays important roles in pollen formation, possibly by regulating the activity of mitochondrial F(1)F(0)-ATP synthase in Arabidopsis pollen grains.