Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes ( PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase β-subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling. Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in seedlings affected in AT5G08670, the gene of the β-subunit of mitochondrial ATP synthase than in wild-type (WT) seedlings upon treatment with lincomycin (LIN) and norflurazon (NF). Further studies showed that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in AT5G08670 mutant seedlings treated with LIN. These changes might be associated with the repression of some transcriptional factors (TFs), such as ARF3/ETTIN ( ETT), PLASTID TRANSCRIPTION FACTOR 1 ( PTF1), CYCLOIDEA AND PCF TRANSCRIPTION FACTOR 2 ( TCP2), and KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1 ( KTF1). These findings indicate that the activity of mitochondrial ATP synthase affects plastid retrograde signaling.