The female gametophyte is crucial for sexual reproduction of higher plants, yet little is known about the molecular mechanisms underlying its development. Here, we report that Arabidopsis thaliana NOP10 (AtNOP10) is required for female gametophyte formation. AtNOP10 was expressed predominantly in the seedling and reproductive tissues, including anthers, pollen grains, and ovules. Mutations in AtNOP10 interrupted mitosis of the functional megaspore during early development and prevented polar nuclear fusion in the embryo sacs. AtNOP10 shares a high level of amino acid sequence similarity with Saccharomyces cerevisiae (yeast) NOP10 (ScNOP10), an important component of the H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) implicated in 18S rRNA synthesis and rRNA pseudouridylation. Heterologous expression of ScNOP10 complemented the mutant phenotype of Atnop10. Thus, AtNOP10 influences functional megaspore mitosis and polar nuclear fusion during gametophyte formation in Arabidopsis.