In the face of increased water scarcity as a major consequence of climate change, improving water use efficiency (WUE) becomes crucial for sustaining viticulture in the Mediterranean area. Leeway to improve WUE depends on how water losses are coupled with biomass gain. As water loss at night is not associated with any carbon gain, its reduction could be a good strategy to limit waste of water without altering photosynthesis rate in the daytime. However, genetic and physiological bases of variations in night transpiration are still unclear. The present study aimed at elucidating whether and how variations in night transpiration are genetically controlled in grapevine. A 2 year experiment was conducted on the F1 progeny from a cross between Syrah and Grenache using a phenotyping platform coupled to a controlled-environment chamber. Potted plants of the whole progeny were analysed for night transpiration, both in well-watered and water deficit conditions. A high genetic variability was found and 7 QTLs were detected, of which 3 were stable between years. These results evidenced for the first time the genetic control of water losses at night by identifying underlying locus. We then explored the physiological bases of this genetic variability, deciphering the respective roles of stomata and cuticle. 20 genotypes, chosen within the progeny for their contrasting transpirations at night, were studied in experiments with detached leaves. By feeding leaves with abscisic acid, we showed that stomata remain open at night, and that this residual opening differs among genotypes. We took advantage of the hypostomatic feature in grapevine to study cuticular conductance on the adaxial face. We found a significant genetic control of cuticular conductance, which accounted for up to 30% of the total leaf transpiration at night. These results open new avenues to breed grapevine for lower water losses at night, whose genetic variability appears to be driven both by stomata and cuticle.