Nearly 3 billion additional urban dwellers are forecasted by 2050, an unprecedented wave of urban growth. While cities struggle to provide water to these new residents, they will also face equally unprecedented hydrologic changes due to global climate change. Here we use a detailed hydrologic model, demographic projections, and climate change scenarios to estimate per-capita water availability for major cities in the developing world, where urban growth is the fastest. We estimate the amount of water physically available near cities and do not account for problems with adequate water delivery or quality. Modeled results show that currently 150 million people live in cities with perennial water shortage, defined as having less than 100 L per person per day of sustainable surface and groundwater flow within their urban extent. By 2050, demographic growth will increase this figure to almost 1 billion people. Climate change will cause water shortage for an additional 100 million urbanites. Freshwater ecosystems in river basins with large populations of urbanites with insufficient water will likely experience flows insufficient to maintain ecological process. Freshwater fish populations will likely be impacted, an issue of special importance in regions such as India's Western Ghats, where there is both rapid urbanization and high levels of fish endemism. Cities in certain regions will struggle to find enough water for the needs of their residents and will need significant investment if they are to secure adequate water supplies and safeguard functioning freshwater ecosystems for future generations.