This dataset contains concentrations of 27 polar metabolites measured in gill and mantle tissue of individual European flat oysters (Ostrea edulis) sampled before the start of the chronic alkalinity-enhancement exposure (7 May 2024, baseline, n = 10 per tissue) and after 21, 42 and 63 days of exposure (28 May, 18 June and 9 July 2024) to one of five treatments, with 3 to 5 individuals sampled per treatment, tissue and timepoint. Extraction: Frozen gill and mantle samples were wet-weighed (100 to 400 mg WW) and, if necessary, ground to smaller pieces with a mortar and pestle pre-chilled in liquid nitrogen before being transferred to Precellys tubes. Polar metabolites were extracted following the protocol of Tripp-Valdez et al. (2017), adjusted as follows: 400 µL methanol and 125 µL MilliQ water for homogenization and 400 µL chloroform and 400 µL MilliQ water for phase separation. All steps used pre-chilled (0 to 4 °C) instruments and reagents to minimize metabolite degradation. The polar upper layer was transferred to 1.5 mL tubes and dried overnight in a SpeedVac vacuum concentrator at 30 °C (RVC 2-18, Martin Christ Freeze Dryers GmbH, Germany). Dried polar metabolites were resuspended in D2O containing 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP; 0.05 wt %; Sigma Aldrich, St. Louis, USA) as internal standard and chemical shift reference, in a 1:1 ratio of µL D2O per mg tissue WW. Measurement: Untargeted metabolite profiling was performed using an ultra-shielded vertical 9.4 T NMR spectrometer (Avance III HD 400 WB, Bruker-BioSpin GmbH, Germany) equipped with a 1.7 mm TXI MicroProbe. Metabolite spectra were acquired using TopSpin 3.2 (Bruker-BioSpin GmbH, Rheinstetten, Germany) at room temperature, with the following parameters: 1D-Carr-Purcell-Meiboom-Gill (CPMG) pulse train with f1 presaturation (cpmgpr1d), 90° pulse 8.4 µs, TD 70.656, sweep width 8802 Hz (22 ppm), acquisition time 4.01 s, relaxation delay 4 s, 4 dummy scans and 128 scans per sample. ¹H-NMR spectra were processed and analyzed in Chenomx NMR Suite V.12.0 (Chenomx Inc., Canada): automated zero-filling, exponential multiplication (0.3 Hz line broadening), phase correction, baseline correction, shim correction and TSP calibration. Spectra from each tissue type within experimental groups were overlaid in Chenomx Profiler to evaluate spectral quality, comparability, linewidths, shifts and baselines, a step critical for accurate metabolite quantification. The adenylates ATP, ADP and AMP were summed as one metabolite (adenylates), resulting in a total of 27 metabolites.