In plants, water use efficiency is a complex trait arising from numerous physiological and developmental characteristics. Here, we investigated the involvement of circadian regulation in long-term water use efficiency in Arabidopsis under light and dark conditions. Circadian rhythms are generated by the circadian oscillator, which provides a cellular measure of the time of day. In plants, the circadian oscillator contributes to the regulation of many aspects of physiology, including stomatal opening, rate of photosynthesis, carbohydrate metabolism, and developmental processes such as the initiation of flowering. We investigated the impact of the misregulation of numerous genes encoding various components of the circadian oscillator on whole plant, long-term water use efficiency. From this analysis, we identified a role for the circadian oscillator in water use efficiency. It appears that the circadian clock contributes to the control of transpiration and biomass accumulation. We also established that the circadian oscillator within guard cells can contribute to long-term water use efficiency. Our experiments indicate that knowledge of circadian regulation will be important for developing crops with improved water use efficiency.