Human TRF1-binding protein PinX1 inhibits telomerase activity. Here we report that overexpression of yeast PinX1p (yPinX1p) results in shortened telomeres and decreased in vitro telomerase activity. yPinX1p coimmunoprecipitated withyeast telomerase protein Est2p even in cells lacking the telomerase RNA TLC1, or the telomerase-associated proteins Est1p and Est3p. Est2p regions required for binding to yPinX1p or TLC1 were similar. Furthermore, we found two distinct Est2p complexes exist, containing either yPinX1p or TLC1. Levels of Est2p–yPinX1p complex increased when TLC1 was deleted and decreased when TLC1 was overexpressed. Hence, we propose that yPinX1p regulates telomerase by sequestering its protein catalytic subunit in an inactive complex lacking telomerase RNA.