Nighttime transpiration has been previously reported as a significant source of water loss in many species; however, there is a need to determine if this trait plays a key role in the response to drought. This study aimed to determine the magnitude, regulation and relative contribution to whole plant water-use, of nighttime stomatal conductance (g(night)) and transpiration (E-night) in grapevine (Vitis vinifera L.). Our results showed that nighttime water loss was relatively low compared to daytime transpiration, and that decreases in soil and plant water potentials were mainly explained by daytime stomatal conductance (g(day)) and transpiration (E-day). Contrary to E-day, E-night did not respond to VPD and possible effects of an innate circadian regulation were observed. Plants with higher g(night) also exhibited higher daytime transpiration and carbon assimilation at midday, and total leaf area, suggesting that increased g(night) may be linked with daytime behaviors that promote productivity. Modeling simulations indicated that g(night) was not a significant factor in reaching critical hydraulic thresholds under scenarios of either extreme drought, or time to 20% of soil relative water content. Overall, this study suggests that g(night) is not significant in exacerbating the risk of water stress and hydraulic failure in grapevine.