AbstractSprayingN-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), an exogenous cytokinin (CK) growth regulator, is the conventional method for inducing fruit set during melon (Cucumis meloL.) production; however, the mechanism by which CPPU induces fruit set is unclear. Through histological and morphological observations, fruit size was comparable between CPPU-induced fruits and normal pollinated fruits because CPPU-induced fruits had higher cell density but smaller cell size compared with normal pollinated fruits. CPPU promotes the accumulation of gibberellin (GA) and auxin and decreases the level of abscisic acid (ABA) during fruit set. Moreover, application of the GA inhibitor paclobutrazol (PAC) partially inhibits CPPU-induced fruit set. Transcriptome analysis revealed that CPPU-induced fruit set specifically induced the GA-related pathway, in which the key synthase encodinggibberellin 20-oxidase 1(CmGA20ox1) was specifically upregulated. Further study indicated that thetwo-component response regulator 2(CmRR2) of the cytokinin signaling pathway, which is highly expressed at fruit setting, positively regulates the expression ofCmGA20ox1. Collectively, our study determined that CPPU-induced melon fruit set is dependent on GA biosynthesis, providing a theoretical basis for the creation of parthenocarpic melon germplasm.