We present one millennium-long (1171-year), and three 100 year long annually resolved δ13C tree-ring chronologies from ecologically varying Juniperus stands in the Karakorum Mountains (northern Pakistan), and evaluate their response to climatic and atmospheric CO2 changes. All δ13C records show a gradual decrease since the beginning of the 19th century, which is commonly associated with a depletion of atmospheric δ13C due to fossil fuel burning. Climate calibration of high-frequency δ13C variations indicates a pronounced summer temperature signal for all sites. The low-frequency component of the same records, however, deviates from long-term temperature trends, even after correction for changes in anthropogenic CO2. We hypothesize that these high-elevation trees show a response to both climate and elevated atmospheric CO2 concentration and the latter might explain the offset with target temperature data. We applied several corrections to tree-ring δ13C records, considering a range of potential CO2 discrimination changes over the past 150 years and calculated the goodness of fit with the target via calibration/verification tests (R2, residual trend, and Durbin–Watson statistics). These tests revealed that at our sites, carbon isotope fixation on longer timescales is affected by increasing atmospheric CO2 concentrations at a discrimination rate of about 0.012‰/ppmv. Although this statistically derived value may be site related, our findings have implications for the interpretation of any long-term trends in climate reconstructions using tree-ring δ13C, as we demonstrate with our millennium-long δ13C Karakorum record. While we find indications for warmth during the Medieval Warm Period (higher than today’s mean summer temperature), we also show that the low-frequency temperature pattern critically depends on the correction applied. Patterns of long-term climate variation, including the Medieval Warm Period, the Little Ice Age, and 20th century warmth are most similar to existing evidence when a strong influence of increased atmospheric CO2 on plant physiology is assumed.