Nocturnal water use (Qnight) is an important component of the eucalyptus water budget, but it has always been under-appreciated and poorly understood. To improve the accuracy of water balance estimates and understanding of the nocturnal water use process in eucalypts plantations, we conducted a 3-year study to investigate the characteristics of Qnight and its components in a Eucalyptus urophylla × E.grandis plantation in southern China. The results showed that the Qnight of E.urophylla × E.grandis was substantial and its contribution (Rnight) to daily water use (Qdaily) was on average 12.35%, with higher Rnight (14.97%) in the dry season than in the wet season (9.50%). However, the Qnight was used not only for nocturnal transpiration (Tn), but also for stem refilling (Re). Tn was influenced by a combination of vapor pressure deficit (VPD), air temperature (Ta) and relative humidity (RH), with VPD being the dominant driver. Based on this, combined with the fact that Re was closely related to diurnal variations in diameter, we have developed a novel method to distinguish Tn from Re. We found that the compositional ratios of Tn and Re differed between weather conditions and months. However, on a 3-year average, Qnight of E.urophylla × E.grandis was still mainly used for Tn (58.63%). Our results highlight the non-ignorability of Qnight and the high variability of the compositional ratios of Re and Tn, and suggest that Qnight and its components should be accurately quantified and taken into account when studying the water balance in eucalyptus stands.