The ecophysiological response of an alpine grassland to recent climate change and increasing atmospheric CO2 concentration was investigated with a new strategy to go back in time: using a time-series of Capra ibex horns as archives of the alpine grasslands' carbon isotope discrimination (13Δ). From the collection of the Natural History Museum of Bern, horns of 24 males from the population of the Augstmatthorn–Brienzer Rothorn mountains, Switzerland, were sampled covering the period from 1938 to 2006. Samples were taken from the beginning of each year-ring of the horns, representing the beginning of the horn growth period, the spring. The horns' carbon 13C content (Δ13C) declined together with that of atmospheric CO2 over the 69-year period, but 13Δ increased slightly (+0.4‰), though significantly (P<0.05), over the observation period. Estimated intercellular CO2 concentration increased (+56 μmol mol−1) less than the atmospheric CO2 concentration (+81 μmol mol−1), so that intrinsic water-use efficiency increased by 17.8% during the 69-year period. However, the atmospheric evaporative demand at the site increased by approximately 0.1 kPa between 1955 and 2006, thus counteracting the improvement of intrinsic water-use efficiency. As a result, instantaneous water-use efficiency did not change. The observed changes in intrinsic water-use efficiency were in the same range as those of trees (as reported by others), indicating that leaf-level control of water-use efficiency of grassland and forests followed the same principles. This is the first reconstruction of the water-use efficiency response of a natural grassland ecosystem to last century CO2 and climatic changes. The results indicate that the alpine grassland community has responded to climate change by improving the physiological control of carbon gain to water loss, following the increases in atmospheric CO2 and evaporative demand. But, effective leaf-level water-use efficiency has remained unchanged.