Climate change and human activity such as reservoir operation have altered the hydrological system in the transboundary Mekong River basin (MRB) over decades, urging a need to assess the historical changes and future projections of freshwater availability. Here we examined changes of terrestrial water storage anomalies (TWSA) from the Gravity Recovery and Climate Experiment satellites in the lower MRB during 2003–2020, and subsequently partitioned and attributed them into climate-driven and non-climate-driven components using the WaterGAP hydrological model (WGHM) with and without consideration of human activities, together with a statistical method driven by climatic forcing only. Further, integrated future TWSA was projected under different climate change scenarios during 2030–2099 forced with four downscaled and bias-corrected simulations of four global climate models. Results show a decreasing TWSA trend of −3.7 ± 1.8 mm/a during 2003–2020. The WGHM-based climate-driven TWSA, which is highly correlated with the statistical modeling results, and non-climate-driven part suggests a trend of −0.3 ± 1.4 and 0.01 ± 0.07 mm/a during 2003–2016, respectively. The climate-driven TWSA is well explained by the changes in decreasing precipitation (−1.3 ± 8.5 mm/a) and increasing air temperature (0.05 ± 0.02 ℃/a) spatially and temporally, while the non-climate-driven component is closely linked to human activities such as growing sectoral human withdrawal (0.13 ± 0.14 mm/a), increasing reservoir regulation (0.01 ± 0.08 mm/a), and changing land cover. TWSA under future climate changes is projected to increase from 9.3 ± 21.4 to 12.2 ± 12.2 mm and from 1.6 ± 41.2 to 12.3 ± 30 mm in the near (2040–2059) and far future (2080–2099) under various scenarios comparing with the historical period (2003–2020). Future flood potential, estimated with TWSA and precipitation, was also projected to increase. This study provides important inferences for decision-makers and stakeholders to better understand the water cycle and manage water resources in a changing environment.