The growth factor heregulin (HRG) promotes breast cancer (BC) tumorigenesis and metastasis and differentially modulates BC cell responses to DNA-damaging agents via its dual extracellular and nuclear localization. Given the central role of telomere dysfunction to drive carcinogenesis and to alter the chemotherapeutic profile of transformed cells, we hypothesized that an unanticipated nuclear function of HRG might be to regulate telomere length. Engineered overexpression of the HRG beta 2 isoform in non-aggressive, HRG-negative MCF-7 BC cells resulted in a significant shortening of telomeres (up to 1.3 kb) as measured by Southern blotting of telomere terminal restriction fragments. Conversely, antisense-mediated suppression of HRG beta 2 in highly aggressive, HRG-overexpressing MDA-MB-231 and Hs578T cells increased telomere length up to 3.0 kb. HRG beta 2 overexpression promoted a marked upregulation of telomere-binding protein 2 (TRF2) protein expression, whereas its knockdown profoundly decreased TRF2 expression. Double staining of endogenous HRG beta 2 with telomere-specific peptide nucleic acid probe/fluorescence in situ hybridization (PNA/ FISH) revealed the partial localization of HRG at the chromosome ends. Moreover, a predominantly nucleoplasmic staining pattern of endogenous HRG beta 2 appeared to colocalize with TRF2 and, concomitantly with RAP1, a telomere regulator that specifically interacts with TRF2. Small interfering RNA-mediated knockdown of HRG decreased the expression of TRF2 and RAP1, decreased their presence at chromosome ends, and coincidentally resulted in the formation of longer telomeres. This study uncovers a new function for HRG beta 2 in controlling telomere length, in part due to its ability to regulate and interact with the telomere-associated proteins TRF2 and RAP1.