The volatile plant hormone ethylene regulates many plant developmental processes and stress responses. It is therefore crucial that plants can precisely control their ethylene production levels in space and time. The ethylene biosynthesis pathway consists of two dedicated steps. In a first reaction, S-adenosyl-L-methionine (SAM) is converted into 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC-synthase (ACS). In a second reaction, ACC is converted into ethylene by ACC-oxidase (ACO). Initially, it was postulated that ACS is the rate-limiting enzyme of this pathway, directing many studies to unravel the regulation of ACS protein activity, and stability. However, an increasing amount of evidence has been gathered over the years, which shows that ACO is the rate-limiting step in ethylene production during certain dedicated processes. This implies that also the ACO protein family is subjected to a stringent regulation. In this review, we give an overview about the state-of-the-art regarding ACO evolution, functionality and regulation, with an emphasis on the transcriptional, post-transcriptional, and post-translational control. We also highlight the importance of ACO being a prime target for genetic engineering and precision breeding, in order to control plant ethylene production levels.