To examine whether the epigenetic status of the human Oct4 promoter is similarly regulated in mouse cells, we engineered a human bacterial artificial chromosome to express green fluorescent protein under the control of the hOct4 promoter and stably integrated it into mouse embryonic stem cells (mESCs), NIH3T3, and 293T cells. The hOct4 promoter is unmethylated in mESCs and it undergoes methylation during retinoic acid-induced differentiation. However, the hOct4 promoter is demethylated in NIH3T3 cells even though it is fully methylated in 293T cells. Methylation status of the hOct4 promoter is associated with green fluorescent protein expression at transcription level. Our findings indicate that the hOct4 promoter is differently regulated in mouse cells.