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The Qaidam Basin, located on the northeastern Tibetan Plateau and containing Cenozoic sediments with a maximum thickness of ~ 12,000 m, is an ideal place to study the phased uplift of the NE Tibetan Plateau and regional climate change. The estimation of the paleosalinity of sedimentary environments not only helps to evaluate the evolution of lakes in this region but offers insights into contemporaneous climate change. We present detailed geochemical and mineralogical investigations from the lacustrine interval of the Hongliugou section in the northern Qaidam Basin to reconstruct salinity fluctuations in the paleolake during the late Eocene era (~ 42.0–35.5 Ma). The clay mineral assemblages mainly contain smectite, illite, chlorite, kaolinite and irregular illite/smectite mixed layers. Clay boron-derived paleosalinity estimates (equivalent boron content, Couch's paleosalimeter and B/Ga ratios) along with other proxies sensitive to salinity changes (e.g., Rb/K ratios and ostracod assemblages) collectively indicate an overall brackish sedimentary environment with a higher-salinity period at approximately 40.0–39.2 Ma. This higher-salinity period indicates a more arid environment and is probably related to global cooling. However, the global cooling in late Eocene cannot explain the overall stable long-term salinity pattern, implying that other factors exist. We propose that the migration of the Yiliping depression depocenter in the northern Qaidam and increased orographic rainfall induced by late Eocene tectonic activity at the northern margin of the basin might have partly offset the increase in salinity driven by global cooling. [ABSTRACT FROM AUTHOR]