Understanding the influences of climatic changes on water use efficiency (WUE) of Tibetan alpine meadows is important for predicting their long-term net primary productivity (NPP) because they are considered very sensitive to climate change. Here, we collected wool materials produced from 1962 to 2010 and investigated the long-term WUE of an alpine meadow in Tibet on basis of the carbon isotope values of vegetation (δ13Cveg). The values of δ13Cveg decreased by 1.34‰ during 1962-2010, similar to changes in δ13C values of atmospheric CO2. Carbon isotope discrimination was highly variable and no trend was apparent in the past half century. Intrinsic water use efficiency (Wi) increased by 18 μmol·mol-1 (approximately 23.5%) during 1962-2010 because the increase in the intercellular CO2 concentration (46 μmol·mol-1) was less than that in the atmospheric CO2 concentration (Ca, 73 μmol·mol-1). In addition, Wi increased significantly with increasing growing season temperature and Ca. However, effective water use efficiency (We) remained relatively stable, because of increasing vapor pressure deficit. Ca, precipitation, and growing season temperature collectively explained 45% of the variation of We. Our findings indicate that the We of alpine meadows in the Tibetan Plateau remained relatively stable by physiological adjustment to elevated Ca and growing season temperature. These findings improve our understanding and the capacity to predict NPP of these ecosystems under global change scenarios.