Abstract P27Kip1 (CDKN1B) regulates cellular proliferation and senescence, and p27Kip1 deficiency in cancer is strongly correlated with poor prognosis of multiple cancer types. Understanding the mechanism of p27Kip1 loss in cancer and the consequences of restoring p27Kip1 levels is therefore critical for effective management during therapy. Here, SIRT1, a class III histone deacetylase (HDAC), is identified as an important regulator of p27Kip1 expression. Mechanistically, SIRT1 reduces p27Kip1 expression by decreasing p27Kip1 protein stability through the ubiquitin–proteasome pathway. In addition, SIRT1 silencing suppresses non–small cell lung cancer (NSCLC) proliferation and induces senescence in a p27Kip1-dependent manner. Furthermore, SIRT1 silencing dramatically suppresses tumor formation and proliferation in two distinct NSCLC xenograft mouse models. Collectively, these data demonstrate that not only SIRT1 is an important regulator of p27Kip1 but also SIRT inhibition induces senescence and antigrowth potential in lung cancer in vivo. Implications: SIRT1 is a key regulator of p27 protein levels and SIRT1 inhibition is a viable strategy for NSCLC therapy by means of p27 reactivation. Mol Cancer Res; 13(1); 41–49. ©2014 AACR.