Tropical rainforest regions are urbanizing rapidly, yet the role of emerging metropolises in driving wildlife overharvesting in forests and inland waters is unknown. We present evidence of a large defaunation shadow around a rainforest metropolis. Using interviews with 392 rural fishers, we show that fishing has severely depleted a large-bodied keystone fish species, tambaqui (Colossoma macropomum), with an impact extending over 1,000 km from the rainforest city of Manaus (population 2.1 million). There was strong evidence of defaunation within this area, including a 50% reduction in body size and catch rate (catch per unit effort). Our findings link these declines to city-based boats that provide rural fishers with reliable access to fish buyers and ice and likely impact rural fisher livelihoods and flooded forest biodiversity. This empirical evidence that urban markets can defaunate deep into rainforest wilderness has implications for other urbanizing socioecological systems.