The Nullarbor Plain in southern Australia is an extensive treeless area (ca. 200,000 km2), almost devoid of surface water, reflecting the modern expression of Plio-Pleistocene aridification. The rapid climatic shift from the humid late Pliocene to arid early Pleistocene drove the Nullarbor Plains development as an important biogeographic barrier, facilitating distinct diversification of numerous taxa to the east and west. Despite the significance of this environmental change, absolute age constraints on terrestrial aridification are rare. To improve understanding on the timing of aridification in the Nullarbor Plain, we combine petrographic, mineralogical, chemical, and (U-Th)/He thermochronological data from ferruginous indurations that overprinted middle-upper Eocene Ooldea Sands in the eastern Nullarbor Plain. Integrated analyses reveal (i) secondary pedogenetic relic features hosted in marine siliciclastic sediments, (ii) later geochemical transformation of pedogenetic features, and (iii) subsequent growth of polycrystalline iron oxides in response to an inferred groundwater decline. Thermochronological results tie the latest stage of this polyphase genesis (i.e., the ferruginization) to the Plio-Pleistocene transition. Review of other local and regional climate proxies shows profound temporal correlations with the formation of ferruginous indurations. These dates constrain a turning point in southern Australia’s landscape evolution, associated with the initiation of a vast arid landscape separating mesic environments, with implications for habitat fragmentation and species diversity.