IIIG9 is the regulatory subunit 32 of protein phosphatase 1 (PPP1R32), a key phosphatase in the regulation of ciliary movement. IIIG9 localization is restricted to cilia in the trachea, fallopian tube, and testicle, suggesting its involvement in the polarization of ciliary epithelium. In the adult brain, IIIG9 mRNA has only been detected in ciliated ependymal cells that cover the ventricular walls. In this work, we prepared a polyclonal antibody against rat IIIG9 and used this antibody to show for the first time the ciliary localization of this protein in adult ependymal cells. We demonstrated IIIG9 localization at the apical border of the ventricular wall of 17-day-old embryonic (E17) and 1-day-old postnatal (PN1) brains and at the level of ependymal cilia at 10- and 20-day-old postnatal (PN10–20) using temporospatial distribution analysis and comparing the localization with a ciliary marker. Spectral confocal and super-resolution Structured Illumination Microscopy (SIM) analysis allowed us to demonstrate that IIIG9 shows a punctate pattern that is preferentially located at the borders of ependymal cilia in situ and in cultures of ependymocytes obtained from adult rat brains. Finally, by immunogold ultrastructural analysis, we showed that IIIG9 is preferentially located between the axoneme and the ciliary membrane. Taken together, our data allow us to conclude that IIIG9 is localized in the cilia of adult ependymal cells and that its expression is correlated with the process of ependymal differentiation and with the maturation of radial glia. Similarly, its particular localization within ependymal cilia suggests a role of this protein in the regulation of ciliary movement.