The gaseous hormone ethylene controls a variety of physiological processes in horticultural plants, including fruit ripening and elongation, flower development and senescence, and responses to stresses. The functions of ethylene in these processes are intimately linked to its precise biosynthesis, which is finely tuned by a complex network of positive and negative regulators. While significant progress has been made in understanding the roles of positive regulators in ethylene biosynthesis, the negative regulators of ethylene biosynthesis has only recently begun to receive more focus. Ethylene biosynthesis is a simple two-step reaction in land plants, committed by two dedicated enzymes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO). Over the past decade, a growing number of research has identified a wide range of transcriptional, posttranscriptional and epigenetic negative regulators for ACS and/or ACO in horticultural plants, greatly enhancing our understanding of the intricate network that modulates ethylene production. In this review, we provide a comprehensive overview of the negative regulators that mediate ethylene biosynthesis in horticultural plants, with respect to their functions and molecular mechanisms, and their responses to external environmental stimuli or internal growth signals.