Abstract: To investigate the patterns and origins of ethylene release in Penicillium digitatum-citrus interaction, ethylene biosynthesis was analyzed both during the infection process and across distinct infection zones (green sporulation zone, white mycelial zone, macerated zone, and healthy zone) by gas chromatography. Additionally, the content of 1-aminocyclopropane-1-carboxylic acid (ACC) and the expression levels of ACC oxidase genes (ACO1 and ACO2) were measured in different infection zones. Citrus were treated with the plant ethylene biosynthesis inhibitor α-aminooxyacetic acid (AOA), as well as the fungal ethylene precursors α-ketoglutaric acid (α-KG) and 2-keto-4-methylthiobutyric acid (KMBA), to determine the ethylene level. The results showed that ethylene production significantly increased during P. digitatum-citrus interaction, characterized by an initial increase followed by a sharp decline. All infected zones accumulated significantly higher ethylene levels compared to the healthy zone, with the most pronounced accumulation in the green sporulation zone. Following the pharmacological inhibition of ethylene biosynthesis in citrus by AOA, ethylene production was partially suppressed. The highest ACC accumulation was observed in the green sporulation zone, while the expressions of ACO1 and ACO2 were nearly completely inhibited. Application of KMBA or α-KG resulted in markedly enhanced ethylene emission and accelerated disease development. In conclusion, ethylene production is significantly enhanced during the P. digitatum-citrus interaction, with varying contributions from different infection zones. Ethylene produced during infection originates from both the host fruit and the pathogen, with the fungal-derived ethylene functioning as a virulence factor. These findings provide new insights to understand the possible pathogenic mechanisms of P. digitatum.