CD45 has been reported to regulate the HIV-1 gp120-induced apoptosis of Jurkat cells. Here, we demonstrate that the extracellular domain of CD45 plays an important role in this function. We observed that CD45RO-transfected cells, but not cells transfected with other CD45 isoforms, underwent significant apoptosis induced by gp120. However, a CD45RA-transfected cell line treated with an O-glycan inhibitor was able to undergo apoptosis. The role of the extracellular domain of CD45 was further confirmed using CD45 isoform-transfected cell lines by analyzing the phosphorylation of Lck, which is a direct substrate of CD45 tyrosine phosphatase, and by using an Lck inhibitor. These results suggest that CD45RO modulates HIV-1 gp120-induced apoptosis by regulating the activity of Lck.