In higher plants, chloroplast and mitochondrial transcripts contain a number of group II introns that need to be precisely spliced before translating into functional proteins. However, the mechanism of splicing and the factors involved in this process is not well understood. By analyzing a seed mutant in maize, we report here the identification of Empty pericarp16 (Emp16) that is required for nad2 intron 4 splicing in mitochondria. Disruption of Emp16 function causes embryo and endosperm development arrest, giving rise to an empty pericarp phenotype in maize. Differentiation of the basal endosperm transfer layer cells is severely affected. Molecular cloning indicates that Emp16 encodes a P-type PPR protein with 11 PPR motifs and is localized in the mitochondrion. Transcript analysis revealed that the mitochondrial nad2 intron 4 splicing is abolished in the emp16 mutants, which leads to severely reduced complex I assembly and activity. In response, the mutant dramatically increases the accumulation of mitochondrial complex III and the expression of alternative oxidase AOX2. These results implicate that EMP16 is specifically required for the mitochondrial nad2 intron 4 cis-splicing, essential for complex I assembly, and the embryogenesis and endosperm development in maize. This article is protected by copyright. All rights reserved.