The mechanisms of root hair formation have been studied extensively in Arabidopsis but knowledge about these processes in monocot species is still limited, especially in relation to the proteome level. The aim of this study was to identify the proteins that are involved in the initiation and the early stage of root hair tip growth in barley using two-dimensional (2D) electrophoresis and mass spectrometry. A comparison of proteins that accumulate differentially in two root hair mutants and their respective parent varieties resulted in the identification of 13 proteins that take part in several processes related to the root hair morphogenesis, such as the control of vesicular trafficking, ROS signalling and homeostasis, signal transduction by phospholipids metabolism and ATP synthesis. Among the identified proteins, two ATP synthases, two ABC transporters, a small GTPase from the SAR1 family, a PDI-like protein, a monodehydroascorbate reductase, a C2 domain-containing protein and a Wali7 domain-containing protein were found. This study is the first report on the proteins identified in the initial stage of root hair formation in barley and gives new insights into the mechanisms of root hair morphogenesis in a monocot species.