The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulators with important roles in replication and transcription. Although regarded as a transcription elongator for chromatin template, little is known about the mode of action and involvement in other molecular processes of the mammalian FACT. Here we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, approximately 600-kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr(210)). When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr(210). Cell cycle analysis on the Nek9(dsRNAi) cells directly implicated Nek9 in maintaining proper G(1) and S progression, a role temporally correlated to the formation of a phospho-Nek9-FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.