Korean fir is an endemic ornamental tree species facing population decline in Korea. To further understand the acclimatory adjustments it undergoes in response to seasonal extremes, we characterized some of the needle proteins that are upregulated during winter. Two-dimensional gel electrophoresis (2-DE), followed by MALDI TOF/TOF MS/MS and Mascot analyses, was used to visualize changes in protein profiles during acclimation to winter stress. From the 2-DE protein profiles of Korean fir needles, 226 protein spots were detected, many of which accumulated at higher levels during the winter. Among 17 proteins identified, 12 matched proteins associated with photosynthesis and with biotic and abiotic stresses, and eight were significantly upregulated under winter stress. Upregulated proteins included photosynthetic enzymes sedoheptulose-1,7-bisphosphatase and fructose bisphosphate aldolase of the Calvin–Benson cycle, four proteins related to oxidative stress tolerance, two proteins implicated in biotic defense, one heat-shock protein, and five unknown proteins. However, two other oxidative-stress-related proteins were present at high levels throughout the year, and a chitinase and the small subunit of ribulose-1,5-bisphosphate carboxylase showed no seasonal adjustments. Thus, Korean fir needles exhibited winter upregulation of some photosynthetic enzymes, coupled with increased photo protective thermal energy dissipation, and proteins related to abiotic and biotic stress resistance. Winter stress, which can include both low temperature and reduced water availability, in the subalpine region of Mount Halla led to an altered physiological equilibrium with increases in key Calvin–Benson cycle enzymes and increased enzymatic and non-enzymatic protection against oxidative stress.