Understanding the impacts of atmospheric [CO2] and drought on leaf respiration (R) and its response to changes in temperature is critical to improve predictions of plant carbon-exchange with the atmosphere, especially at higher temperatures. We quantified the effects of [CO2]-enrichment (+240 ppm) on seasonal shifts in the diel temperature response of R during a moderate summer drought in Eucalyptus saligna growing in whole-tree chambers in SE Australia. Seasonal temperature acclimation of R was marked, as illustrated by: (1) a downward shift in daily temperature response curves of R in summer (relative to spring); (2)≈60% lower R measured at 20oC (R20) in summer compared with spring; and (3) homeostasis over 12 months of R measured at prevailing nighttime temperatures. R20, measured during the day, was on average 30–40% higher under elevated [CO2] compared with ambient [CO2] across both watered and droughted trees. Drought reduced R20 by≈30% in both [CO2] treatments resulting in additive treatment effects. Although [CO2] had no effect on seasonal acclimation, summer drought exacerbated the seasonal downward shift in temperature response curves of R. Overall, these results highlight the importance of seasonal acclimation of leaf R in trees grown under ambient- and elevated [CO2] as well as under moderate drought. Hence, respiration rates may be overestimated if seasonal changes in temperature and drought are not considered when predicting future rates of forest net CO2 exchange.