Ascorbate oxidase (AO, EC 1.10.3.3) is a member of the multicopper oxidases family. It catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbate (MDHA), with the concomitant reduction of molecular oxygen to water. In melon (Cucumis melo), ascorbate oxidase is encoded by a multigene family comprising at least four genes. Here, we present the detailed characterization of two melon AO genes, CmAO1 and CmAO4. Gene-specific expression studies of the AO gene family in melon revealed that only CmAO1 and CmAO4 are transcriptionally active and differentially regulated dependent on tissue, developmental stage and external stimuli. Transcripts of the CmAO1 gene are present in floral and fruit tissues, whereas CmAO4 mRNA preferentially accumulates in vegetative tissues. CmAO genes were not detected in melon seeds, but CmAO4 expression is activated upon germination. CmAO4 mRNA steady-state levels are also regulated in response to wounding and heat stress, by hormones (abscisic acid, salicylic acid and jasmonates), AA and copper. These findings suggest that AO gene expression is transcriptionally regulated during fruit development and in response to hormonal cues associated with the control of cell growth and the stress response.