Human-induced global changes are altering several components of the chemical composition of the atmosphere including a global increase in the ozone concentration of the troposhere. Ozone is a highly reactive gas with strong effects on plant function. One such effect reported in many experiments is a decrease in leaf-level stomatal conductance. If a change in conductance leads to reductions in transpiration at the whole-plant or ecosystem level, the indirect influence of ozone could be large. To investigate this issue, we used a controlled laboratory experiment investigating the mechanistic relationship between ozone and plant transpiration. We fumigated two aspen (Populus tremuloides) genotypes varying in ozone resistance with a 70 ppb ozone enhancement 4 hours per day in open-top chambers for 8 weeks. Transpiration rates were measured using lysimetry and stomatal conductance was quantified using a portable plant gas-exchange system. Over the course of the experiment, whole plant transpiration in the resistant and sensitive genotypes decreased 20 and 30%, respectively, compared to control plants. The influence of ozone exposure increased through time with the largest decreases in transpiration observed after five weeks of fumigation. These results suggest elevated ozone concentrations have the ability to alter ecosystem-level water fluxes and change several important aspects of ecosystem and atmospheric function.