AbstractIsoprene is a C5 volatile organic compound, which can protect aboveground plant tissue from abiotic stress such as short‐term high temperatures and accumulation of reactive oxygen species (ROS). Here, we uncover new roles for isoprene in the plant belowground tissues. By analysing Populus x canescens isoprene synthase (PcISPS) promoter reporter plants, we discovered PcISPS promoter activity in certain regions of the roots including the vascular tissue, the differentiation zone and the root cap. Treatment of roots with auxin or salt increased PcISPS promoter activity at these sites, especially in the developing lateral roots (LR). Transgenic, isoprene non‐emitting poplar roots revealed an accumulation of O2− in the same root regions where PcISPS promoter activity was localized. Absence of isoprene emission, moreover, increased the formation of LRs. Inhibition of NAD(P)H oxidase activity suppressed LR development, suggesting the involvement of ROS in this process. The analysis of the fine root proteome revealed a constitutive shift in the amount of several redox balance, signalling and development related proteins, such as superoxide dismutase, various peroxidases and linoleate 9S‐lipoxygenase, in isoprene non‐emitting poplar roots. Together our results indicate for isoprene a ROS‐related function, eventually co‐regulating the plant‐internal signalling network and development processes in root tissue.