The Eocene-Oligocene climate transition (EOCT) at similar to 34 Ma is a pronounced environmental reorganization that occurred in the Cenozoic era and has been extensively studied via marine archives. However, to date, there is still a less well-dated continental record across the EOCT, which can mostly be attributed to a lack of suitable proxies used in the varied continental settings in terms of sedimentary facies, provenance and climate, thus resulting in a poor understanding of the continental response to this transition. Located on the northeastern Tibetan Plateau, the Qaidam Basin is filled with continuous and well-exposed Cenozoic fluvio-lacustrine sediments. Cenozoic lacustrine sediment has been proven to be an ideal archive to study the continental climatic response to the EOCT. Here, we present the detailed mineralogical and elemental composition of chlorite, a widespread clay mineral in continental sediments formed by rock weathering, collected from a lacustrine sequence in the northern Qaidam Basin to constrain the onset of the EOCT and the related climatic response. The results show that chlorite abundance, crystallinity indices, intensity ratios of chlorite diagnostic peaks and chemical composition collectively exhibit remarkable changes at similar to 34.3 Ma. To be more specific, the well crystallized Fe-Mg-rich chlorites were characterized by a high Fe content before the EOCT and turned into high-Mg-content Mg-chlorites with poor crystallinity. The chlorite chemical composition change is assumed to have been related to regional drying after the EOCT, thus providing new insights into the continental paleoenvironmental change based on this novel proxy.