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Guishui River Basin in northwestern Beijing has ecological significance and will be one of the venues of the upcoming Beijing Winter Olympic Games in 2022. However, accelerating climate change and human disturbance in recent decades has posed an increasing challenge to the sustainable use of water in the basin. This study simulated the runoff of the Guishui River Basin using the Soil and Water Assessment Tool (SWAT) model to reveal the spatio-temporal variations of runoff in the basin and the impacts of climate change and human activities on the runoff changes. The results showed that annual runoff from 2004 to 2018 was relatively small, with an uneven intra-annual runoff distribution. The seasonal trends in runoff showed a decreasing trend in spring and winter while an increasing trend in summer and autumn. There was a first increasing and then decreasing trend of average annual runoff depth from northwest to southeast in the study area. In addition, the contributions of climate change and human activities to changes in runoff of the Guishui River Basin were 60% and 40%, respectively, but with opposite effects. The results can contribute to the rational utilization of water resources in the Guishui River Basin.