RbAp48 is a WD-40 protein that plays an important role in chromatin metabolism and regulates Ras signaling. Here, we report that RbAp48 is involved in the regulation of cytoskeletal organization, a novel function. First, we show that transfection of RbAp48 into Hs-578T breast cancer cells (Hs-RbAp48-hi) leads to cell size reduction, a rounded cell shape, decreased cellular protrusions, and a higher nuclear/cytoplasmic ratio. Furthermore, we observed cytoskeletal F-actin organization disruption with loss of actin stress fibers and formation of membranous F-actin rings in Hs-RbAp48-hi cells. These morphologic changes were partially reversed by RbAp48 knockdown. Interestingly, mitogen-activated protein kinase (MAPK) was activated in Hs-RbAp48-hi cells, and this activity was also partly reversed by RbAp48 down-regulation. Furthermore, pharmacologic inhibition of MAPK led to the reappearance of organized actin fibers and focal contacts, suggesting MAPK as the effector pathway. Moreover, we show an increase in total Ras activity in Hs-RbAp48-hi cells with K-Ras-GTP becoming the dominant isoform. This reverted to baseline activity levels on RbAp48 small interfering RNA transfection, thus suggesting a direct role for RbAp48 in Ras regulation. Finally, we tested the model in transformed 3T3-K-Ras-G12V fibroblasts. As expected, RbAp48 knockdown in 3T3-K-Ras-hi fibroblasts resulted in reappearance of an organized cytoskeleton and shutdown of K-Ras activity. In conclusion, our data support a model whereby RbAp48 regulates cellular morphology and cytoskeletal organization by increasing K-Ras activity and signaling through MAPK.