As one of the most dynamic aspects of global environmental change, land cover change (LCC) has a profound impact on terrestrial carbon sequestration. However, LCC-induced carbon fluxes are still the most uncertain terms in global and regional carbon budgets. Ecosystem gross primary production (GPP) is the total carbon uptake by vegetation through photosynthesis, serving as a major control on ecosystem function and land carbon balance during and after the modification of the land surface. However, accurately capturing LCC-induced GPP changes requires both high-quality land cover data and controlling for variation driven by other environmental factors such as climate. In this study, we comprehensively examined the effects of LCC on annual GPP trends over the conterminous United States (CONUS) from 2001 to 2016 using the USGS National Land Cover Database, a remote sensing-driven ecosystem model, and the Google Earth Engine cloud computing platform. We designed a series of model experiments to identify LCC effects on GPP by controlling climate effects. During the study period, LCC exerted a strong negative effect on total GPP across the CONUS ([-2.2, -1.8] Tg C yr−2), while climate had smaller positive effects ([0.17, 0. 92] Tg C yr−2). The LCC-induced reduction of GPP was mainly caused by net forest loss ([-1.98, -1.39] Tg C yr−2) and urban expansion ([-2.03, -1.92] Tg C yr−2), but was partially offset by increases in crop area ([+0.66, +0.79] Tg C yr−2). Ensemble simulations from TRENDY did not capture the strong negative LCC influences on GPP, likely due to limitations of the adopted land use/cover data. Our study provides a novel perspective on LCC-induced GPP changes, which could help to improve our understanding of ecosystem function changes and constrain the estimation of land carbon balance in the context of anthropogenic activity and climate change.