1. Invasive non-native species (INNS) threaten biodiversity and ecosystem functioning globally. However, there remains a pressing need to understand the environmental factors controlling the dispersal, successful establishment and subsequent ecological impacts of INNS for receiving ecosystems. Here, we examine how region-wide flow regime magnitudes facilitate the successful establishment of an invasive crayfish species (Pacifastacus leniusculus, signal crayfish) in England (UK). We also consider the interactive effects of invasive crayfish with flow regime variations on the structural and functional diversity of macroinvertebrate communities. 2. Low-flow magnitudes increased the likelihood of P. leniusculus establishment, with 80% of recorded invasion dates falling in years with flow magnitudes below average (low-and low-moderate flow classes), whilst only 1.6% occurred in high-flow years. 3. Temporal trajectories of structural and functional macroinvertebrate responses in invaded rivers demonstrated reduced diversity compared to control rivers. Lower taxonomic and functional richness measures typically coincided with periods of low discharge in invaded rivers and were greatest during regionally high-flows. 4. Macroinvertebrate communities displayed significant structural and functional responses to the interaction between invasive crayfish and flow regime variations.Specifically, a number of low-and high-flow indices yielded significant associations, highlighting the role of extreme hydrological events in shaping INNS effects on receiving ecosystems. We also detected greater ecological effects of invasive crayfish under hydrologically stable conditions. Importantly, and for the first time, we observed that invasive crayfish reversed macroinvertebrate community responses to flow regime cues (e.g. discharge fall rate and minimum flows in the preceding 180 days). 5. Synthesis and applications. Results from this study indicate that low-flow events facilitate the spread/establishment of invasive crayfish and correspond with greater ecological effects for receiving ecosystems. Given that low-flow events are predicted to increase in intensity, duration and frequency over the 21st century, our results highlight the potential threat that invasive crayfish may pose under future hydroclimatic changes. Managing river flow regimes effectively (including | Additional supporting information may be found online in the Supporting Information section. How to cite this article: Mathers KL, White JC, Fornaroli R, Chadd R. Flow regimes control the establishment of invasive crayfish and alter their effects on lotic macroinvertebrate communities. J Appl Ecol. 2020;57:886-902. https://doi.