Flows provide cues for a range of critical life history behaviours in fishes such as movement and spawning. In many rivers throughout the world, however, flow regulation has resulted in the loss or disruption of these cues. The provision of environmental flows provides an important opportunity to reinstate these cues to assist fish population recovery. A major challenge of environmental flows science and management therefore is developing understanding of critical relationships between flow regimes and fish behavioural responses. The Australian grayling is a diadromous fish species that has declined considerably since European settlement. Changes to flow regimes are a major reason for the decline. Consequently, environmental flow recommendations have been developed and implemented for the species in many regulated rivers. Although such efforts may benefit Australian grayling, gaps in knowledge of flow-ecology relationships limit the development and effectiveness of these strategies. To address this shortcoming, we investigated two critical life history behaviours of Australian grayling (movement and spawning) and relationships with flows, using larval drift sampling and acoustic telemetry. In particular, the study tested whether Australian grayling migrate to specific areas to spawn, and whether these behaviours are influenced by flow. The study found that Australian grayling migrate large distances downstream to spawn coinciding with increased river flows. The study demonstrates the need to understand fish behaviour and life history to develop appropriate flow regimes for fish in regulated rivers. Importantly, this information is now being used by river managers to refine existing environmental flow recommendations for the species.