The development of effective strategies to restore the biological functioning of aquatic ecosystems with altered flow regimes requires a detailed understanding of flow-ecology requirements, which is unfortunately lacking in many cases. By understanding the flow conditions required to initiate critical life history events such as migration and spawning, it is possible to mitigate the threats posed by regulated river flow by providing targeted environmental flow releases from impoundments. In this study, we examined the influence of hydrological variables (e.g., flow magnitude), temporal variables (e.g., day of year) and spatial variables (e.g., longitudinal position of fish) on two key life history events (migration to spawning grounds and spawning activity) for a threatened diadromous fish (Australian grayling Prototroctes maraena) using data collected from 2008 to 2015 in the Bunyip–Tarago river system in Victoria. Our analyses revealed that flow changes act as a cue to downstream migration, but movement responses differed spatially: fish in the upper catchment showed a more specific requirement for rising discharge to initiate migration than fish in the lower catchment. Egg concentrations peaked in May when weekly flows increased relative to the median flow during a given spawning period. This information has recently been incorporated into the development of targeted environmental flows to facilitate migration and spawning by Australian grayling in the Bunyip–Tarago river system and other coastal systems in Victoria.