Freshwater is key to Kenya’s socioeconomic development but also essential for maintaining environmental integrity and human welfare. Here, we report the results of a comprehensive and collaborative assessment of the ecological status of the Mara River and the environmental flow regime needed to sustain the ecological function of the river and related systems in the Mara/Serengeti Ecoregion. Field surveys indicate only modest evidence of degradation based on the geomorphology of the river channel and the presence and vitality of select indicator riparian plants, fish, and macroinvertebrates. Environmental flow recommendations were based on hydraulic modelling and field observations and were determined through a consensus building process involving members of the research team. During normal rainfall years, recommendations range from average monthly values of 1.3 to 15.0m3 s−1. During drought years, the respective recommended flows are 0.3 to 6.0m3 s−1. These recommendations fall between the 45th percentile of monthly flow durations curve during wet months of normal years and the 98th percentile of the annual flow duration curves during drought years. Recommended floods range from 2-day events of 12m3 s−1 to an annual 3-day flood of 90m3 s−1. Results of the assessment suggest that during years of normal rainfall there is sufficient flow in the river to allow additional water extractions and still meet environmental requirements, but environmentally friendly storage is needed and uncontrolled abstractions could easily exhaust the available resource. During drought years, however, the river may already drop to flows incapable of simultaneously meeting both extractive water demands and environmental flow recommendations.