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In the Brazilian Amazon, private land accounts for the majority of remaining native vegetation. Understanding how land-use change affects the composition and distribution of biodiversity in farmlands is critical for improving conservation strategies in the face of rapid agricultural expansion. Working across an area exceeding 3 million ha in the southwestern state of Rondônia, we assessed how the extent and configuration of remnant forest in replicate 10,000-ha landscapes has affected the occurrence of a suite of Amazonian mammals and birds. In each of 31 landscapes, we used field sampling and semistructured interviews with landowners to determine the presence of 28 large and medium sized mammals and birds, as well as a further 7 understory birds. We then combined results of field surveys and interviews with a probabilistic model of deforestation. We found strong evidence for a threshold response of sampled biodiversity to landscape level forest cover; landscapes with <30-40% forest cover hosted markedly fewer species. Results from field surveys and interviews yielded similar thresholds. These results imply that in partially deforested landscapes many species are susceptible to extirpation following relatively small additional reductions in forest area. In the model of deforestation by 2030 the number of 10,000-ha landscapes under a conservative threshold of 43% forest cover almost doubled, such that only 22% of landscapes would likely to be able to sustain at least 75% of the 35 focal species we sampled. Brazilian law requires rural property owners in the Amazon to retain 80% forest cover, although this is rarely achieved. Prioritizing efforts to ensure that entire landscapes, rather than individual farms, retain at least 50% forest cover may help safeguard native biodiversity in private forest reserves in the Amazon.