Maintenance of telomere is regulated by active telomerase complex, including telomerase holoenzyme and its associated proteins. The activity of telomerase is precisely controlled in cells, and its dysregulation is one of the hallmarks of cancer. The telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) plays a central role for telomerase activity. In this study, we indentified that Polo-like kinase 1 (Plk1) is a novel telomerase-associated protein. Plk1 can interact with hTERT independently of its kinase activity. More importantly, we found that Plk1 is associated with active telomerase complex. In addition, we demonstrated that knockdown of Plk1 caused the reduction of telomerase activity, whereas overexpression of Plk1 increased telomerase activity. Further analysis showed that overexpression of Plk1 led to a significant increase of hTERT protein by prolonging its half-life but did not affect the level of hTERT mRNA. Furthermore, we found that Plk1 enhanced the chromatin loading of hTERT and inhibited its ubiquitination. This implied that Plk1 affected hTERT stability by inhibiting its ubiquitin-mediated degradation. Collectively, these observations suggested that Plk1 is a positive modulator of telomerase by enhancing the stability of hTERT.