The Neoproterozoic Era was punctuated by the ‘Sturtian’ (~710million years ago) and ‘Marinoan’ (~635million years ago) low-latitude glaciations. Carbonates preceding the younger Marinoan glacial succession record an ~18‰ negative shift in the δ¹³C of carbonate around the world. This ‘Trezona’ isotopic anomaly is the largest δ¹³C shift in Earth history and its origin and timing remain controversial. The δ¹³C anomaly could record a dramatic reorganization of Earth's carbon cycle and be linked causally to the initiation of Marinoan ice-house conditions. Alternatively, the δ¹³C anomaly might record secondary fluid alteration following carbonate deposition. Here we document dropstones within the carbonate sediments immediately below the Marinoan glacial diamictite in South Australia. Advancing ice sheets caused soft-sediment deformation of the beds below the glacial diamictite, as well as subglacial erosion of the carbonates beneath, showing that the Trezona δ¹³C values must have been acquired before glaciation. Although these stratigraphic relationships do not provide a specific mechanism to explain the Trezona δ¹³C anomaly, they do require that the nadir of the Trezona δ¹³C anomaly was recorded prior to local glacier advance and long before late-stage burial diagenesis could have occurred. Furthermore, the δ¹³C recovery in the Trezona Formation toward 0‰ was synchronous with the appearance of icebergs in the tropics.