Antarctic ice microalga Chlamydomonas sp. can thrive undisturbed under high UV radiation in the Antarctic ice layer. However, it is unknown that the initial adaptation mechanisms in protein level occurring in response to high UV radiation. Global-expression profiling of proteins in response to stress was analyzed by two-dimensional electrophoresis (2-DE) and image analysis. In the 2-DE analysis, protein preparation is the key step. Three different protein extract methods were compared, and the results showed that the trichloroacetic acid (TCA)-acetone fractional precipitation method was the fittest one. At the same time, the proteins in Chiamydomonas sp. were compared in 2-DE way, and the synthesis of seven protein spots was found disappeared and 18 decreased after exposed to UV-B radiation. In addition, 14 protein spots were enhanced or induced, among which two new peptides (20 and 21 kDa) appeared whose isoelectric point (pI) was 7.05 and 4.60 respectively. These changed proteins might act as key role in the acclimation of Antarctic ice microalga to UV-B radiation.