Southwest China is an ecologically fragile area with a high proportion of karstland and large variations in terrain, and it can be greatly affected by land use change. From 2000 to 2020, with the acceleration of urbanization in the whole country, the macro-scale Grain-for-Green Program (GFGP) has been developed in the karstland of southwest China. This has resulted in the expansion of forest and construction land with a reduction in cultivated land. The response of ecosystem services (ESs) to these changes needs to be investigated. However, there is a lack of in-depth analysis of the karstland of southwest China, and current studies mostly focus on the spatio-temporal variation in individual ESs or how the whole land use change affects ESs. Thus, our study uses an InVEST model and geographically and temporally weighted regression (GTWR) to examine the spatio-temporal variation in land use associated with four ESs, namely water conservancy (WC), soil conservancy (SC), carbon sequestration (CS), and habitat quality (HQ). We found that the GFGP area first increased and then decreased, aggregating to 4.48 × 104 km2 from 2000 to 2020. We also observed that from 2000 to 2020, ESs remained stable or gradually increased (despite fluctuations), SC was the most stable, whereas WC, CS, and HQ first decreased and then fluctuated more. This may be related to the destruction of topsoil in the early stages of the GFGP and the relatively weak ES supply capacity of the young trees. Moreover, the response of ecosystem services to the GFGP was spatially heterogeneous, suggesting a strong driving influence of the local environment, mainly caused by the distribution of karstland and terrain, differentiated urbanization levels, and the intensity of the GFGP. Specifically, the relatively significantly positive effects of WC, SC, and CS were found in western and northern cities, and so was the HQ in southeastern cities. On the contrary, the negative impacts of urbanization were found to weaken over time, suggesting the trade-off effect of the GFGP. Our findings would contribute to the development of effective forest management strategies and provide valuable insights for policymakers and stakeholders involved in ecosystem restoration and conservation efforts by exploring the impacts of the GFGP and urbanization on ESs.