Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKIα co-purifies with centaurin-α1 in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217-233. These residues are well conserved in all members of the CKI family, and we show that centaurin-α1 associates in vitro with all mammalian CKI isoforms. To date, CKIα represents the first protein partner identified for centaurin-α 1. However, our data suggest that centaurin-α1 is not a substrate for CKIα and has no effect on CKIα activity. Centaurin-α1 has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-α1 contains a cysteine-rich domain that is shared by members of a newly identified family of ADP-ribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKIα in these biological events.