Climate-driven changes in high-elevation forest distribution and reductions in snow and ice cover have major implications for ecosystems and global water security. In the Greater Yellowstone Ecosystem of the Rocky Mountains (United States), recent melting of a high-elevation (3,091 m asl) ice patch exposed a mature stand of whitebark pine (Pinus albicaulis) trees, located ~180 m in elevation above modern treeline, that date to the mid-Holocene (c. 5,950 to 5,440 cal y BP). Here, we used this subfossil wood record to develop tree-ring-based temperature estimates for the upper-elevation climate conditions that resulted in ancient forest establishment and growth and the subsequent regional ice-patch growth and downslope shift of treeline. Results suggest that mid-Holocene forest establishment and growth occurred under warm-season (May-Oct) mean temperatures of 6.2 °C (±0.2 °C), until a multicentury cooling anomaly suppressed temperatures below 5.8 °C, resulting in stand mortality by c. 5,440 y BP. Transient climate model simulations indicate that regional cooling was driven by changes in summer insolation and Northern Hemisphere volcanism. The initial cooling event was followed centuries later (c. 5,100 y BP) by sustained Icelandic volcanic eruptions that forced a centennial-scale 1.0 °C summer cooling anomaly and led to rapid ice-patch growth and preservation of the trees. With recent warming (c. 2000–2020 CE), warm-season temperatures now equal and will soon exceed those of the mid-Holocene period of high treeline. It is likely that perennial ice cover will again disappear from the region, and treeline may expand upslope so long as plant-available moisture and disturbance are not limiting.