MCPH1, a repressor of human telomerase reverse transcriptase (hTERT) function, is implicated in cellular immortalization. But little is known about how MCPH1 represses telomerase activity. In this study, to determine the mechanism by which MCPH1 regulates hTERT gene expression, we examined the role of MCPH1 in regulating the hTERT promoter in vitro. Co-transfection of the hTERT promoter with MCPH1 in Hela cells could inhibit the hTERT promoter activity. The EMSA assay demonstrated that MCPH1 could bind to the proximal hTERT promoter. Overexpression of MCPH1 could repress telomerase activity, and the repression was abolished by knocking down the MCPH1 expression using siRNA in U2OS cells. We propose that MCPH1 functions as a transcriptional repressor of hTERT in vitro. Since the activation of telomerase, widely observed in human tumor cell lines, is a critical step in tumorigenesis, our findings provide new insights into delineating the tumor-suppressing function of MCPH1 through its down-regulation of hTERT/telomerase expression.