One hallmark of tumor formation is the transcriptional upregulation of human telomerase reverse transcriptase, hTERT, and the resultant induction of telomerase activity. However, little is presently understood about how hTERT is differentially activated in tumor cells versus normal somatic cells. Specifically, it is unclear if oncoproteins can directly elicit hTERT expression. To this end, we now show that three oncoproteins, HER2/Neu, Ras, and Raf, stimulate hTERT promoter activity via the ETS transcription factor ER81 and ERK mitogen-activated protein (MAP) kinases. Mutating ER81 binding sites in the hTERT promoter or suppression of ERK MAP kinase-dependent phosphorylation of ER81 rendered the hTERT promoter unresponsive to HER2/Neu. Further, expression of dominant-negative ER81 or inhibition of HER2/Neu significantly attenuated telomerase activity in HER2/Neu-overexpressing SKBR3 breast cancer cells. Moreover, HER2/Neu, Ras, and Raf collaborated with ER81 to enhance endogenous hTERT gene transcription and telomerase activity in hTERT-negative, nonimmortalized BJ foreskin fibroblasts. Accordingly, hTERT expression was increased in HER2/Neu-positive breast tumors and breast tumor cell lines relative to their HER2/Neu-negative counterparts. Collectively, our data elucidated a mechanism whereby three prominent oncoproteins, HER2/Neu, Ras, and Raf, may facilitate tumor formation by inducing hTERT expression in nonimmortalized cells via the transcription factor ER81.