Oct-3 is a key molecule for maintaining self-renewal in mouse embryonic stem (ES) cells. The function of Oct-3 in ES cells of other species, however, especially primate ES cells, is not clear. In the present study, we cloned two splicing isoforms of Oct-3, Oct-3A and Oct-3B, from cynomolgus monkey ES cells, and found that they have high homology to human Oct-3A and Oct-3B. To examine their function, Oct-3A and Oct-3B were overexpressed in cynomolgus monkey ES cells. Transient Oct-3A overexpression induced ES cell differentiation into endodermal and mesodermal lineages and disrupted proliferation of undifferentiated monkey ES cells. In contrast, Oct-3B overexpression did not induce differentiation of monkey ES cells. These findings indicate that a certain Oct-3A expression level has an important role in sustaining self-renewal in non-human primate ES cells.