Global climate models project more frequent drought events in Central China. However, the effect of seasonal drought on ecosystem water use efficiency (WUE) and water regulation strategy in Central China’s natural forests is poorly understood. This study investigated variations in WUE associated with drought in a natural oak (Quercus aliena) forest in Central China from 2017 to 2020 at several timescales based on continuous CO2 and water vapor flux measurements. Results showed that the 4-year mean gross ecosystem production (GEP), evapotranspiration (ET) and WUE of the natural oak forest was 1613.2 ± 116 g Cm−2, 637.8 ± 163.3 mm and 2.6 ± 0.68 g Ckg−1 H2O, with a coefficient of variation (CV) of 7.2%, 25.6% and 26.4%, respectively. The inter-annual variation in WUE was large, primarily due to the variation in ET caused by seasonal drought. Drought increased WUE distinctly in summer and decreased it slightly in autumn. During summer drought, surface conductance (gs) usually decreased with an increase in VPD, but the ratios of stomatal sensitivity (m) and reference conductance (gsref) were 0.21 and 0.3 molm−2s−1ln(kPa)−1 in the summer of 2019 and 2020. Strong drought can also affect ecosystem WUE and water regulation strategy in the next year. Decrease in precipitation in spring increased annual WUE. These results suggested that drought in different seasons had different effects on ecosystem WUE. Overall, our findings suggest that the natural oak forest did not reduce GEP by increasing WUE (i.e., reducing ET) under spring and summer drought, which could be due to its typical anisohydric characteristics, although it can also reduce stomatal opening during long-term drought.