Seasonal drought events induced by climate change have exacerbated the water deficit in trees. This is particularly pronounced in mature trees with considerable heights and extensive branches, where the long water transport distance leads to a reduction in hydraulic transportation efficiency. Therefore, investigating the water uptake patterns of mature trees is essential to enhance their growth and resilience to climate change. However, previous studies have predominantly focused on trees aged 10–60 years, with limited research on the water uptake patterns of trees over 60 years old. In this study, we employed hydrogen and oxygen stable isotopes coupled with the MixSIAR model to determine the water uptake patterns of mature Quercus aliena var. acuteserrata (40–60 years, 60–90 years, 120–150 years, >150 years) during the growing seasons of 2021 and 2022 in Baotianman Nature Reserve, Henan Province, China. Additionally, we utilized a random forest model to quantify the relative contributions of vegetation (fine root biomass) and soil properties (soil moisture, bulk density, total porosity, field capacity, and soil texture) to the water uptake patterns of Q. aliena. Our findings demonstrate that most mature Q. aliena predominantly extracted deep soil water (60–100 cm) in the early growing season, but shifted towards utilizing shallow soil water (0–40 cm) in the late growing season. The water uptake pattern of mature Q. aliena was primarily regulated by soil moisture. These results reveal that mature Q. aliena trees exhibit a flexible water use strategy, enabling them to cope with seasonal drought by altering the soil depth from which they extract water. In the future, in the process of the cultivation and conservation of mature oak trees, surface soil irrigation can be increased to prevent tree mortality resulting from water deficit during drought conditions.