Elevated atmospheric CO2 adversely affects freezing tolerance in many evergreens, but the underlying mechanism(s) have been elusive. We compared effects of elevated CO2 with those of daytime warming on acclimation of snow gum (Eucalyptus pauciflora) to freezing temperatures under field conditions. Reduction in stomatal conductance gc under elevated CO2 was shown to cause leaf temperature to increase by up to 3 °C. In this study, this increase in leaf temperature was simulated under ambient CO2 conditions by using a free air temperature increase (FATI) system to warm snow gum leaves during daytime, thereby increasing the diurnal range in temperature without affecting temperature minima. Acclimation to freezing temperatures was assessed using measures of electrolyte leakage and photosynthetic efficiency of leaf discs exposed to different nadir temperatures. Here, we show that both elevated CO2 and daytime warming delayed acclimation to freezing temperatures for 2–3 weeks after which time freeze tolerance of the treated plants in both the FATI and open top chamber (OTC) experiments did not differ from control plants. Our results support the hypothesis that delayed development of freezing tolerance under elevated CO2 is because of higher daytime leaf temperatures under elevated CO2. Thus, potential gains in productivity in response to increasing atmospheric CO2 and prolonging the growing season may be reduced by an increase in freezing stress in frost-prone areas.