Eucalyptus grandis is the most widely used eucalypt plantation species due to its rapid growth and superior wood quality. Recent progress in eucalyptus improvement has been possible by the use of biotechnology. Among the molecular tools necessary to make further progress in this area, the identification and characterization of promoters with specific organ/tissue expression patterns is a priority. In this context, the present study aimed to functionally characterize the promoter region of an E. grandis gene encoding a high affinity potassium transporter (named EgHAK) showing root-specific expression. The corresponding EST was selected by in silico predictions in the Eucalyptus EST project (FORESTs) database and its tissue specificity was validated using quantitative PCR (qPCR). An expression cassette containing an EgHAK promoter::GUS fusion was obtained and inserted into tobacco plants. In these plants, GUS staining was exclusively detected in the vascular tissues of leaves and roots. The expression in vascular bundles was confirmed in histological cross sections. To evaluate the promoter responsiveness to low potassium concentrations, two distinct transgenic tobacco lines (T2) were submitted to potassium starvation, and the relative expression of the GUS reporter gene was determined by qPCR. As a result, an increase in the relative expression of GUS in response to potassium starvation was observed.