Reservoirs have altered the flow regime of most rivers on the globe. To simulate the natural flow regime, experimental floods are being implemented on regulated rivers throughout the world to improve their ecological integrity. As a large-scale disturbance, the long-term sequential use of floods provides an excellent empirical approach to examine ecosystem regime shifts in rivers. This study evaluated the long-term effects of floods (15 floods over eight years) on a regulated river. We hypothesized that sequential floods over time would cause a regime shift in the ecosystem. The floods resulted in little change in the physicochemistry of the river, although particulate organic carbon and particulate phosphorus were lower after the floods. The floods eliminated moss cover on bed sediments within the first year of flooding and maintained low periphyton biomass and benthic organic matter after the third year of flooding. Organic matter in transport was reduced after the third year of flooding, although peaks were still observed during rain events due to tributary inputs and side slopes. The floods reduced macroinvertebrate richness and biomass after the first year of floods, but density was not reduced until the third year. The individual mass of invertebrates decreased by about one-half after the floods. Specific taxa displayed either a loss in abundance, or an increase in abundance, or an increase followed by a loss after the third year. The first three flood years were periods of nonequilibrium with coefficients of variation in all measured parameters increasing two to five times from those before the floods. Coefficients of variation decreased after the third year, although they were still higher than before the floods. Analysis of concordance using Kendall's W confirmed the temporal changes observed in macroinvertebrate assemblage structure. An assessment of individual flood effects showed that later floods had approximately 30% less effect on macroinvertebrates than early floods of similar magnitude, suggesting that the new assemblage structure is more resilient to flood disturbance. We conclude that the floods caused an ecosystem regime shift that took three years to unfold. Additional long-term changes or shifts are expected as new taxa colonize the river from other sources.