Aberrations in cell cycle control are often observed in tumors and might even be necessary in tumor development. Spy1, a novel cell cycle regulatory protein, can control cell progression and survival through the atypical activation of cyclin-dependent kinases (CDKs). In this progression, the phosphorylation of p27(Kip1) at Thr187 by CDK2 was shown to be a chief role. In this study, we studied 183 human specimens including reactive lymphoid and Non-Hodgkin's Lymphomas (NHLs) tissues. Immunohistochemistry (IHC) analysis suggested that Spy1 and pThr187-p27 were overexpressed in NHLs. The expression of Spy1 was positively related to pThr187-p27 and proliferation marker Ki-67 expression. In a multivariate analysis, high Spy1 and pThr187-p27 expressions were showed to be associated with poor prognosis in NHLs. While in vitro, following release of Jurkat cells from serum starvation, the expression of Spy1 was upregulated, as well as pThr187-p27 and CDK2. And an increased interaction between Spy1 and pThr187-p27 was demonstrated at 4 h after serum stimulation. Additionally, transfecting cells with Spy1-siRNA could diminish the expression of pThr187-p27 and arrest cell growth. Our results suggest that Spy1 may be a possible prognostic indicator in NHLs, and it was correlated with phosphorylation of p27(Kip1) on Thr187. These findings provide a rational framework for further development of Spy1 inhibitors as a novel class of anti-tumor agents.