The discovery of the ancient city of Loulan in Xinjiang, China, at the beginning of the 20th century was of great significance for understanding the evolution of culture and civilization in Inner Asia. However, due to the lack of systematic chronological studies, the history of this ancient city remains unclear, particularly the date of its construction and abandonment. Here, we present the results of the first systematic radiocarbon (14C) dating carried out on artifacts from ancient Loulan. Our results show that human activity began as early as 350 cal BC, flourished during the interval from the 1st to 4th centuries AD, and completely disappeared around 600 AD. Most of the buildings in the city were constructed during the Eastern Han Dynasty rather than in Wei/Jin Dynasty, as previously indicated by excavated documents and letters (Hedin 1898; Xiao 2006). The development and flourishing of Loulan coincided with the interval of high ice accumulation and meltwater supply from surrounding mountains. The city began to decline and was finally abandoned following an abrupt decrease in ice accumulation and meltwater supply (Yao et al. 1996; Lauterbach et al. 2014), suggesting that natural climate change was the major factor responsible for the abandonment of Loulan.