Octamer-binding factor 3/4 (Oct3/4) is one of the key regulators maintaining the pluripotency and self-renewal in embryonic stem cells and is involved in the developmental events. However, the functional significance of Oct3/4 remains to be clarified during tooth morphogenesis. This study aimed to examine the functional role of Oct3/4 in mouse. During tooth morphogenesis (E11-E16.5), Oct3/4-positive cells, detected by nuclear immunoreaction, increased in number, and subsequently, their immunoreaction shifted from the nucleus to the cytoplasm at the stage of cell differentiation (E18.5). Quantitative real-time PCR clearly demonstrated the relationship between isoforms of Oct3/4 and the in vivo cellular localization of Oct3/4, suggesting that the Oct3/4 expressed in nucleus was Oct3/4A, whereas that expressed in the cytoplasm was Oct3/4B. RNAi knockdown of Oct3/4 induced apoptosis and arrested tooth morphogenesis. Our results suggest that (1) the increased number of Oct3/4-positive cells with nuclear immunoreaction correlate with active cell proliferation during tooth morphogenesis and (2) the shift of Oct3/4 from the nucleus to the cytoplasm plays a crucial role in cell differentiation.