The factor PinX1 has been shown as a telomerase inhibitor evolutionarily conserved in both the yeast and the human being. yPinX1 inhibits telomerase activity by sequestering yTERT (telomerase reverse transcriptase) from uniting with yTR (telomerase template RNA) in the nucleolus of yeast cells. However, the mechanism underlying the action of hPinX1 on telomerase regulation in human cells is not known. We here demonstrated that hPinX1 actually has an effect on mediating hTERT nucleolar localization and this effect is mediated by a novel domain enclosed within the central section of the polypeptide. Interestingly, we showed that a reported cancerous mutant form of hPinX1, in which residues of Ser254 and Cys265 are, respectively, mutated to Cys and Tyr, lost the activity on mediating hTERT nucleolar localization. Finally, we provided evidence that mediation of hTERT nucleolar localization and telomerase enzymatic inhibition are two separated function of hPinX1 on telomerase regulation in human cells.