Variations in glacier meltwater in the source regions of the Tibetan Plateau's (TP) largest lake (Selin Co) and China's longest river (Yangtze River) regulate the local and downstream water balance under the warming climate. However, the magnitude of their variations over the past four decades is still unknown. Here, we examine long-term changes in glacier mass balance over 1976–2017 using KH-9 and CoSSC-TanDEM-X data. We find that the mean rate of glacier mass loss (GML) has accelerated from −0.21 ± 0.11 m a–1 over 1976–2000, to −0.28 ± 0.14 m a–1 over 2000–11, and subsequently to −0.48 ± 0.10 m a–1 over 2011–17. Changes in temperature and precipitation are the major causes of GML. Over 1976–2017, the contribution of decadal GML to Tuotuohe sub-basin runoff ranges from 4.3 to 8.0%, while its contribution to increased lake volume change in Selin Co and Chibzhang Co-Dorsoidong Co ranges from 3.5 to 16.3% and 19.2 to 21.4%, respectively. The GML of source regions made relatively small contributions to river runoff and lake supply, but plays a vital role when precipitation decreases. The quantitative evaluation of the water balance for the sources of great rivers and lakes over the TP is therefore important for water resource management and hydrological cycle studies.