Quaternary stable isotope records of marine and lacustrine carbonate deposits as well as speleothems were extensively studied to reconstruct global and regional climatic evolution. This study demonstrates how stable isotope records of travertine provide fundamental information about climate and the consequences of its evolution on groundwater level fluctuations. The deposition of the Lapis Tiburtinus travertine succession occurred during the Late Pleistocene (150–30 ka), coeval with the last activity of the Colli Albani volcanic complex. Two boreholes (Sn1 and Sn2) were drilled into the Acque Albule Basin (23 km E of Rome), crossing the entire Lapis Tiburtinus succession. The Sn1 borehole in the central part of the basin crosscuts a travertine succession of 62.1 m in thickness, while the Sn2 borehole in the southern part of the basin is characterized by a travertine succession 36.3 m in thickness. Carbon and oxygen stable isotope ratios were analysed on 118 samples (59 samples both for Sn1 and Sn2 boreholes) representative of the entire Lapis Tiburtinus travertine succession crossed by the boreholes. Values, measured and correlated in the two drilled boreholes, permitted determination of the sensitivity of the travertine depositional system to glacial and interglacial cycles, unravelling the complex oxygen and carbon cycle dynamic recorded in such sedimentary succession. Moreover, the results obtained correlated with available pollen curves of the Mediterranean area (from the Castiglione crater, 25 km E of Rome). Regional and global oxygen isotope continental and marine curves, calibrated with the stratigraphy of the Acque Albule Basin, and available U/Th dating allow the identification of at least three phases of the last interglacial (Marine Isotope Stage 5-MIS5). The carbon isotope record, compared with CO2 flux reconstructed and associated with the volcanic activity of the Colli Albani volcanic complex, instead shows an influence from groundwater level changes. In particular, positive shifts that occurred during arid phases are associated with a lower groundwater level and increased CO2 degassing, inducing a major fractionation effect on carbon isotopes. Instead, the negative shifts occurring during more humid periods indicate the inhibition of CO2 degassing and increase in pressure, attesting to a rise in groundwater level. In this view, travertine deposits, frequently studied to define the tectonic setting and activity of the area where they develop, can thus also be used as a tool to understand climate changes and groundwater variations apparent in their stable oxygen and carbon isotope signature.