The water conservation service of an ecosystem reflects the sustainability of regional water resources and is significant to human survival and sustainable development. However, global climate warming and intensified human activities pose substantial challenges to regional water conservation services, especially in an alpine region with a fragile ecological environment, which is more sensitive to climate factors and land use pattern changes. In this study, the Qilian Mountains (QLM) region was chosen as the study area to investigate water conservation trends and drivers in an alpine region. The InVEST model was used to estimate water conservation in the QLM from 2000 to 2020. In addition, the characteristics of the spatiotemporal variation in the water conservation were analyzed using a combination of the Theil–Sen median trend and Mann–Kendall method, coefficient of variation, and Hurst exponent, and the main driving factors affecting these changes were determined using partial correlation analysis and contribution analysis. The main conclusions are as follows: (1) The predicted water conservation in the QLM based on the InVEST model’s water yield module had a relative inaccuracy of 5.96%, and the mean water conservation in the QLM from 2000 to 2020 was approximately 78.08 × 108 m3. (2) The water conservation showed a slight increase over the whole QLM region, with a change rate of 0.565 mm/a; yet, it showed a distinct spatial distribution pattern characterized by “more in the east than in the west”. (3) The contribution of the various land use categories to the total water conservation, from highest to lowest, was according to the following: grassland (62.44%) > unutilized land (15.99%) > forest (11.44%) > cultivated land (9.86%) > construction land (0.45%) > water (0.03%). (4) Precipitation exhibited a significant positive correlation, with contribution ratios of approximately 58.50% to the variation in the water content, whereas potential evapotranspiration and surface temperature showed a nonsignificant negative correlation with contribution ratios of approximately 2.17% and 2.08%, respectively. The results can provide scientific reference for ecological protection in the QLM and other similar alpine environment areas.