Net ecosystem productivity (NEP) is an important indicator of the capacity of terrestrial ecosystems to absorb carbon. For the purpose of developing adaptation and mitigation strategies, it is essential to investigate the effect of climatic changes and anthropogenic activities on NEP. This study analyzed the spatiotemporal properties of NEP in the Yangtze River and Yellow River Source Region (SRYY) using remote sensing images, climate variable data, and land use data. Then it was examined at how climatic change affected carbon sinks and sources and how changes in land use affected carbon storage. According to the results, the SRYY contributed a carbon sink worth 35.26 Tg C annually from 2001 to 2019 (1 Tg = 1012 g). Influenced by climate, soil and vegetation, NEP displayed regional differences in space. The spatial distribution of NEP gradually increased from Northwest to Southeast. In recent twenty years, the NEP of SRYY slightly increased with a rate of 3.24 Tg C per decade. The NEP with an increasing trend accounted for 78.9% of the whole study area, which were widely distributed and especially significant in the west, such as Tuotuo River Basin. The correlation analysis between NEP and climate variables suggested that temperature was the dominant control of NEP in the SRYY. About 54.4% of the NEP variance across the entire SRYY was contributed by temperature. However, there was no significant linear relationship between NEP and annual precipitation. In recent two decades, built-up land expansion and ecological degradation reduced carbon sink by 28.61 Gg C (1 Gg = 109 g) and 1.94 Tg C, respectively. Grassland degradation caused by rodent infestation and grazing was the main factor of carbon sink reduction in some areas. The restoration of vegetation and wetland areas resulted in a 0.75 Tg C increase in the carbon sink. It might be benefited from warmer climate condition and ecological protection measures.