Octamer 4 (Oct4), a member of the POU family of transcription factors, plays a key role in the maintenance of pluripotency and proliferation potential of embryonic stem cells. Cancer stem cell–like cells (CSCLC) are reported to be a minor population in tumors or even in tumor cell lines which also express Oct4. The role of Oct4 in CSCLCs still remains to be defined. In our study, we show that, in vitro, almost all murine Lewis lung carcinoma 3LL cells and human breast cancer MCF7 cells express Oct4 at high levels. This expression of Oct4 is successfully reduced by small interfering RNA, which eventually results in cell apoptosis. The signal pathway Oct4/Tcl1/Akt1 has been observed to be involved in this event. The repression of Oct4 reduces Tcl1 expression and further down-regulates the level of p-Ser.473-Akt1. In vivo, only ∼5% of tumor cells were detected to express Oct4 in established 3LL and MCF7 tumor models, respectively. Small interfering RNA against Oct4 successfully decreases the CSCLCs and markedly inhibits tumor growth. In summary, we show that Oct4 might maintain the survival of CSCLCs partly through Oct4/Tcl1/Akt1 by inhibiting apoptosis, which strongly indicates that targeting Oct4 may have important clinical applications in cancer therapy. [Cancer Res 2008;68(16):6533–40]