Coordination of gene expression in the organelles and the nucleus is important for eukaryotic cell function. Transcriptional and post-transcriptional gene regulation in mitochondria remains incompletely understood in most eukaryotes, including plants. Here we show that poly(A)-specific ribonuclease, which influences the poly(A) status of cytoplasmic mRNA in many eukaryotes, directly regulates the poly(A) tract of mitochondrial mRNA in conjunction with a bacterial-type poly(A) polymerase, AGS1, in Arabidopsis. An Arabidopsis poly(A)-specific ribonuclease-deficient mutant, ahg2-1, accumulates polyadenylated mitochondrial mRNA and shows defects in mitochondrial protein complex levels. Mutations of AGS1 suppress the ahg2-1 phenotype. Mitochondrial localizations of AHG2 and AGS1 are required for their functions in the regulation of the poly(A) tract of mitochondrial mRNA. Our findings suggest that AHG2 and AGS1 constitute a regulatory system that controls mitochondrial mRNA poly(A) status in Arabidopsis.