We examined the response of fishes to establishment of a new flow regime in lower Putah Creek, a regulated stream in California, USA. The new flow regime was designed to mimic the seasonal timing of natural increases and decreases in stream flow. We monitored fish assemblages annually at six sample sites distributed over ∼30 km of stream for eight years before and nine years after the new flow regime was implemented. Our purpose was to determine whether more natural stream flow patterns would reestablish native fishes and reduce the abundances of alien (nonnative) fishes. At the onset of our study, native fishes were constrained to habitat immediately (<1 km) below the diversion dam, and alien species were numerically dominant at all downstream sample sites. Following implementation of the new flow regime, native fishes regained dominance across more than 20 km of lower Putah Creek. We propose that the expansion of native fishes was facilitated by creation of favorable spawning and rearing conditions (e.g., elevated springtime flows), cooler water temperatures, maintenance of lotic (flowing) conditions over the length of the creek, and displacement of alien species by naturally occurring high-discharge events. Importantly, restoration of native fishes was achieved by manipulating stream flows at biologically important times of the year and only required a small increase in the total volume of water delivered downstream (i.e., water that was not diverted for other uses) during most water years. Our results validate that natural flow regimes can be used to effectively manipulate and manage fish assemblages in regulated rivers.