The removal of fruit cluster leaves was shown to be a valuable method for controlling fruit quality and producing high-grade apples. A chemical defoliant that functions as an activation switch was shown to turn on the genes necessary for fruit cluster leaf defoliation. Elucidating the mechanism involved in leaf defoliation is crucial to our understanding of the use of chemical defoliants in fruit trees. To gain insight into chemical defoliant-mediated leaf defoliation, we first confirmed the occurrence of ethylene production by applying a chemical defoliant on fruit cluster leaves. Then, we used RNA-seq analysis to obtain a series of transcriptome profiles for genes and proteins involved in leaf senescence induction. Within 2 days of applying the chemical defoliant to apple trees, 1-aminocyclopropane carboxylic acid (ACC) oxidase, ACC synthase, a chlorophyll binding protein, and polygalacturonase-related genes were up-regulated at least sixfold. An in vitro enzyme assay showed that lanolin oil activates ACC synthase activity, a key regulatory enzyme in the ethylene pathway. We also showed that chemical defoliant decreased the light saturation point and total chlorophyll content. Then, we used a polygalacturonase activity assay to confirm the effects of chemical defoliant on leaf senescence in vivo. Furthermore, treatment with chemical defoliant resulted in a significant increase in the chromaticity value of a*, whereas L* and b* decreased in the apple fruit. Taken together, we conclude that chemical defoliant could selectively affect fruit cluster leaves, which suggested that it can be used as a selective defoliant.