In many regions, drought is suspected to be a cause of Scots pine decline and mortality, but the underlying physiological mechanisms remain unclear. Because of their relationship to ecohydrological processes, δ¹⁸O values in tree rings are potentially useful for deciphering long-term physiological responses and tree adaptation to increasing drought. We therefore analyzed both needle- and stem-level isotope fractionations in mature trees exposed to varying water supply. In a first experiment, we investigated seasonal δ¹⁸O variations in soil and needle water of Scots pine in a dry inner Alpine valley in Switzerland, comparing drought-stressed trees with trees that were irrigated for more than 10 years. In a second experiment, we analyzed twentieth-century δ¹⁸O variations in tree rings of the same forest, including a group of trees that had recently died. We observed less ¹⁸O enrichment in needle water of drought-stressed compared with irrigated trees. We applied different isotope fractionation models to explain these results, including the Péclet and the two-pool correction, which considers the ratio of unenriched xylem water in the needles to total needle water. Based on anatomical measurements, we found this ratio to be unchanged in drought-stressed needles, although they were shorter. The observed lower ¹⁸O enrichment in needles of stressed trees was therefore likely caused by increased effective path length for water movement within the leaf lamina. In the tree-ring study, we observed lower δ¹⁸O values in tree rings of dead trees compared with survivors during several decades prior to their death. These lower values in declining trees are consistent with the lower needle water ¹⁸O enrichment observed for drought-stressed compared with irrigated trees, suggesting that this needle-level signal is reflected in the tree rings, although changes in rooting depth could also play a role. Our study demonstrates that long-term effects of drought are reflected in the tree-ring δ¹⁸O values, which helps to provide a better understanding of past tree physiological changes of Scots pine.