Pak1 (p21-activated kinase 1), a conserved, mammalian signaling kinase, is a downstream effector of small GTPases Rac1 and Cdc42 and of growth factor signaling. Until now, a major focus of study has been on the cytosolic functions of Pak1, where it is an important modulator of cytoskeletal reorganization, consequently playing a major role in cell survival, migration, and invasion. In this report, we demonstrate the nuclear localization of Pak1 upon stimulation by epidermal growth factor. Three nuclear localization signals (NLSs) were identified in the N-terminal domain of Pak1. With mutational analysis, the importance of each NLS was elucidated. Mutation of all three NLSs eliminated the nuclear localization of Pak1. Expression of Pak1 as a fusion protein with Gal4-DNA binding domain and Gal4-luciferase activity showed that Pak1 might increase transcription. To identify the potential targets of nuclear Pak1, we used a Pak1-specific chromatin immunoprecipitation-based screening assay and identified a series of Pak1-interacting target chromatins, including phosphofructokinase-muscle isoform (PFK-M) and nuclear factor of activated T-cell (NFAT1) genes. Pak1 associated with the upstream enhancer sequence and promoter of PFK-M and was involved in the stimulation of the PFK-M expression. It also associated with a portion of the NFAT1 gene and its upstream region, leading to the repression of NFAT1 expression. These investigations provide proof-of-principle evidence that Pak1 could influence the expression of its putative chromatin targets in both a positive and a negative manner. Together, for the first time, these findings defined the NLSs of the Pak1, its association with chromatin, and the resulting modulation of transcription, thus opening new avenues to further the search for nuclear Pak1 functions and identify putative Pak1-interacting nuclear proteins.