p21-activated protein kinase 1 (Pak1) plays an important role in several cellular processes, including cytoskeleton reorganization, promotion of the cell survival, and the estrogen receptor (ER) signaling. Pak1 expression and activity is deregulated in a number of cancers. Pak1 is activated by a variety of physiological signals; however, less is known about the negative regulators of Pak1. Here, we report a negative regulator of Pak1. By performing a yeast two-hybrid screen of a mammary gland library, we identified cysteine-rich inhibitor of Pak1 (CRIPak) as a novel Pak1-interacting protein. We found that CRIPak is an intronless gene that localized to chromosome 4p16.3. It contains 13 zinc-finger domains and has three trypsin inhibitor-like, cysteine-rich domains and is widely expressed in a number of human cells and tissues. We further found that CRIPak interacted with Pak1 through the N-terminal regulatory domain and inhibited Pak1 kinase in both in vitro and in vivo assays. CRIPak inhibited Pak1-mediated LIM kinase activation and enhancement of ER transactivation. Conversely, selective inhibition of the endogenous CRIPak resulted in an increased Pak1 activity, and consequently, increased cytoskeleton remodeling and Pak1-mediated ER transactivation activity. The hormonal stimulation of cells enhanced CRIPak expression and promoted its colocalization with ER in the nuclear compartment. Our findings suggest that CRIPak is a novel negative regulator of the Pak1 and has a role in the modulation of Pak1-mediated ER transactivation in breast cancer cells.