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Fruit ripening in climacteric fruit requires the gaseous phytohormone ethylene.Although ethylene signaling has been well studied,knowledge of the transcriptional regulation of ethylene biosynthesis is still limited.Here we show that an apple (Malusdomestica) ethylene response factor,MdERF2,negative affects ethylene biosynthesis and fruit ripening by suppressing the transcription ofMdACS1,a gene that is critical for ripening-related ethylene biosynthesis.MdERF2 expression was suppressed by ethylene during apple fruit ripening,and we observed that MdERF2 bound to the promoter of MdACS1 and directly suppressed its transcription.Moreover,MdERF2 suppressed the activity of the promoter ofMdERF3,a transcription factor that we found to bind to the MdACS1 promoter,thereby increasingMdACS1 transcription.We determined that MdERF2 and MdERF3 proteins directly interact and this interaction suppresses the binding of MdERF3 to the MdACS1 promoter.Moreover,apple fruits with transiently down-regulated MdERF2expression showed higher ethylene production and faster ripening.Our results indicate that MdERF2 negatively affects apple ethylene biosynthesis and fruit ripening by suppressing the transcription of MdACS1via multiple mechanisms,thereby acting as an antagonist of positive ripening regulators.Our findings offer a deep understanding of the transcriptional regulation of ethylene biosynthesis during climacteric fruit ripening.