The aim of this study was to investigate the effects of limited water availability imposed as repeated periods of drought and rehydration on xylem structure and function in stems of a commercially important climber (Humulus lupulus L.). To evaluate drought-induced changes in xylem characteristics, hydraulic measurements, silicone injection, and anatomical observations were performed in basal and apical regions of hop stems. We found that the total measured hydraulic conductivity (Kh) was about 8.5 times lower in the basal regions of drought-treated plants compared to well-watered control plants, especially due to vessel clogging with tyloses. In contrast, Kh of the apical parts of drought-treated plants was higher than in control plants as a result of the enhanced production of secondary xylem. We also observed differences in Kh response to sap ionic composition, with the highest ionic effect in the apical segments of control plants. Greater variation of inner vessel diameter, which increased the transport lumen resistivity of individual vessels by 7%, was another important consequence of irregular watering. The results of our study indicate that periodical fluctuations in water availability induce a significant reduction in total stem transport efficiency and increased risk of vessel dysfunction and illustrate the negative effects of limited water availability for water transport in hop plant stems.