As a significant ecological barrier in northwest China， the Qilian Mountains， it is crucial to clarify the spatial-temporal variation of its ecosystem services （ESs） and evolution mechanism for regional ecological protection and restoration and sustainable development. Using the InVEST （Integrated Valuation of Ecosystem Services and Trade-offs） and CASA （Carnegie-Ames-Stanford Approach） models， we have quantified six critical ESs between 2000 and 2020 including water yield， carbon sequestration， soil retention， nutrient retention， carbon storage， and habitat quality in Qilian Mountains and then analyzed their spatial-temporal variability and spatial distribution characteristics at various scales， and the hotspots of ESs were identified. According to the results， since 2000， the six ESs in Qilian Mountains have been increasing in the region as a whole as well as in different land types， with the mountainous areas being the high value areas and the hotspot areas of category III and IV in the hotspot analysis showing an increasing trend； The six ESs in Qilian Mountains are spatially characterized as being “high in the east and low in the west”， with high values of supply per unit area for forest and grassland， and large differences in the supply capacity of ecosystem services in different topographic gradients， mostly peaking above 3 100 m above sea level， with category V hotspots in the hotspot analysis mainly located in the eastern mountainous areas. The results of the study identify the spatial-temporal variability of six ESs in the Qilian Mountains and explore the natural and anthropogenic factors affecting their spatial and temporal variability， which can provide the basis and suggestions for the formulation of targeted ecological conservation strategies and high-quality sustainable development policies in the Qilian Mountains.