Physcomitrella patens is well known because of its importance in the study of plant systematics and evolution. The tolerance of P. patens for high-salinity environments also makes it an ideal candidate for studying the molecular mechanisms by which plants respond to salinity stresses. We measured changes in the proteome of P. patens gametophores that were exposed to high-salinity (250, 300, and 350 mM NaCl) using two-dimensional gel electrophoresis (2-DE) via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sixty-five protein spots were significantly altered by exposure to the high-salinity environment. Among them, 16 protein spots were down-regulated and 49 protein spots were up-regulated. These proteins were associated with a variety of functions, including energy and material metabolism, protein synthesis and degradation, cell defense, cell growth/division, transport, signal transduction, and transposons. Specifically, the up-regulated proteins were primarily involved in defense, protein folding, and ionic homeostasis. In summary, we outline several novel insights into the response of P. patens to high-salinity; (1) HSP70 is likely to play a significant role in protecting proteins from denaturation and degradation during salinity stress, (2) signaling proteins, such as 14-3-3 and phototropin, may work cooperatively to regulate plasma membrane H+-ATPase and maintain ion homeostasis, (3) an increase in photosynthetic activity may contribute to salinity tolerance, and (4) ROS scavengers were up-regulated suggesting that the antioxidative system may play a crucial role in protecting cells from oxidative damage following exposure to salinity stress in P. patens.