Populations rely on already present plastic responses (ancestral plasticity) and evolution (including both evolution of mean trait values, constitutive evolution, and evolution of plasticity) to adapt to novel environmental conditions. Because of the lack of evidence from natural populations, controversy remains regarding the interplay between ancestral plasticity and rapid evolution in driving responses to new stressors. We addressed this topic at the level of the metabolome utilizing a resurrected natural population of the water flea Daphnia magna that underwent a human-caused increase followed by a reduction in predation pressure within ~16 years. Predation risk induced plastic changes in the metabolome which were mainly related to shifts in amino acid and sugar metabolism, suggesting predation risk affected protein and sugar utilization to increase energy supply. Both the constitutive and plastic components of the metabolic profiles showed rapid, likely adaptive evolution whereby ancestral plasticity and evolution contributed nearly equally to the total changes of the metabolomes. The subpopulation that experienced the strongest fish predation pressure and showed the strongest phenotypic response, also showed the strongest metabolomic response to fish kairomones, both in terms of the number of responsive metabolites and in the amplitude of the multivariate metabolomic reaction norm. More importantly, the metabolites with higher ancestral plasticity showed stronger evolution of plasticity when predation pressure increased, while this pattern reversed when predation pressure relaxed. Our results therefore highlight that the evolution in response to a novel pressure in a natural population magnified the metabolomic plasticity to this stressor. We tested for effects of fish kairomones, subpopulation and their interaction on the metabolomes in a full factorial experiment. The total design consisted of 6 clones × 3 subpopulations × 2 fish kairomone treatments × 8 replicates = 288 experimental units. The metabolome of Daphnia samples were analysed using nano-electrospray ionization - direct infusion mass spectrometry (nESI-DIMS) as described by Southam et al. (2017). Briefly, polar metabolites were first extracted from D. magna samples using a biphasic method. Then, all samples were analysed in both positive and negative ionisation modes using an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) with a direct infusion, chip-based nano-electrospray ionization source (Triversa, Advion Biosciences, Ithaca, NY, USA). The data processing was done using the Galaxy online platform using the selected ion monitoring (SIM) stitching algorithm and data were acquired from mass to charge ratios (m/z) in the range 50–620 (Southam et al., 2017). After several steps (replicate filter, blank filter, sample filter, see details in Supporting Information), the processed data matrices were used for bioinformatics and statistical analyses. Thre are two datasets: one for the positive ionization mode and one for the negative ionization mode. The file type is .raw which is generated from DIMS, and a specific software (not open access) is needed to open the file. We can confirm that these files: 1) do not contain any copywritten or licensed materials, 2) these files do not contain and human subject information or endangered species information, 3) these files are not corrupt. A detailed list of all the files and the meanings for each file was in the ReadMe file.