Peach gummosis caused by Lasiodiplodia theobromae is one of the most detrimental diseases in southern China. This disease is characterized with necrosis and gum exudation in shoots. Ethylene (ET) is known to induce gum formation, however its role in defense against peach fungal gummosis and regulatory mechanism remain unclear. Here, the ET effect on gummosis progression was investigated in peach shoots treated with ET or ET inhibitors before L. theobromae inoculation. Peach shoot infection induced ET production and the transcription of related genes; ET application increased gum formation whereas ET inhibitors repressed it. Additionally, ET treatment significantly reduced jasmonic (JA) level and downregulated OPRs transcript, but increased salicylic acid (SA) content and upregulated PAL expression. ET application increased arabinose content, but decreased glucose and fructose levels. Transcripts of genes related to UDP-sugar metabolism, sugar transporters, and cell wall degradation were upregulated upon ET treatment, and reduced by ET inhibitors. Therefore, ET negatively regulates symptom development in L. theobromae-infected shoots, likely by modulating the JA/SA response and promoting UDP-sugar metabolism and transport, and cell wall degradation during gummosis. Our findings shed light on the molecular mechanism by which ET orchestrates plant defense and gum formation in peach during L. theobromae infection.