Chestnut is an ecologically and economically important species, yet vegetative propagation of mature material remains challenging due to its recalcitrance, limiting biotechnological applications. This study examined adventitious root formation in mature chestnut microshoots, which typically show poor rooting. Silver thiosulphate, an ethylene-signalling inhibitor, was applied prior to auxin induction to assess ethylene’s role. Pretreatment significantly increased rooting, indicating that ethylene negatively affects root induction. Transcriptomic and qPCR analyses suggest this improvement is largely linked to enhanced expression of auxin transport genes, which appear repressed by ethylene. Other hormones may also hinder rooting in mature shoots, though their roles require further clarification.