The scarcity of water resources is becoming a global focus, and water conservation has become one of the most crucial service functions in the water security and sustainable development of ecosystems. Hulunbuir forest–steppe ecotone, as an important water conservation area in the northeastern provinces of China, plays an irreplaceable role in Northeastern China. However, the water yield and water conservation are rarely understood in the ecotone. In this study, the InVEST model was employed to analyze the spatiotemporal dynamics of water yield and water conservation from 2000 to 2020. Meanwhile, we explored the response of water conservation to climatic factors and human disturbance. The results demonstrated that water yield and water conservation presented a decreasing trend in the first decade and then increased. The land use transfer obvious from 2000 to 2010, and most vegetation types were converted into unused land. This transition intensified reduction of water conservation. The main factor affecting the water conservation was climate Precipitation had the greatest impact on water conservation. The findings of this study have great and important implications for regional sustainable water resource management and ecological protection policies and provide a convenient method for evaluating water conservation in other areas that are lacking climate, hydrology, and geological data.