The climatic transition between Eocene and Oligocene (EOT) is marked by the change from a greenhouse to an icehouse climatic mode that affects both the marine and terrestrial realms. Terrestrial ecosystems are more sensitive to climate changes but suffer from the lack of continuous records. Recently, the BRGM retrieved a 340 m-long core from the Rennes Basin, which includes a lacustrine sedimentary series that covers the EOT. In order to decipher the climatic evolution during the EOT and its impacts on ecosystems, we analyzed molecular biomarkers and their isotopic composition. Indices calculated from branched GDGT concentrations allowed us estimating the evolution of mean annual air temperature. The paleohydrological reconstruction relied on the dD of n-alkanes and the Average Chain Length (ACL) of n-alkanes. The evolution of temperature records is marked by two cooling episodes (one during the late Early Priabonian and a second during the Early Rupelian) and warming intervals observed during the Early Priabonian and the EOT. From the Late Priabonian to the Early Rupelian, recurrent ACL and n-alkanes dD values variations indicate changes in relative humidity. One of these variations occurs during the EOT and is interpreted as the transition from humid to dry conditions. The evolution of vegetation is unraveled through specific biomarkers of ferns (fernenes), gymnosperms (diterpenes) and angiosperms (triterpene ketones) and compared with palynological results. The latter shows an increase in mesothermal species from the Late Eocene to the Early Oligocene, in agreement with the global cooling that characterizes this interval. However, the evolution of biomarkers does not show any global trend but high frequency changes from the Late Eocene to the Early Oligocene. These high frequencies are consistent with the rhythm of ACL values. The evolution of continental ecosystems thus appears mainly paced by changes in relative humidity.