Cell-cell contacts mediated by cadherins are known to inhibit the small Rho-GTPase RhoA. We here show that in epithelial cells the disruption of these cell-cell contacts as mediated by a calcium switch leads to actin re-organization and the activation of RhoA. We identified the serine/threonine kinase protein kinase D1 (PKD1) as a downstream target for RhoA in this pathway. After disruption of cell-cell contacts, PKD1 relayed RhoA activation to the induction of the transcription factor NF-κB. We found that a signaling complex composed of the kinases ROCK, novel protein kinase C (nPKC), and Src family kinases (SFKs) is upstream of PKD1 and crucial for RhoA-mediated NF-κB activation. In conclusion, our data suggest a previously undescribed signaling pathway of how RhoA is activated by loss of cell-cell adhesions and by which it mediates the activation of NF-κB. We propose that this pathway is of relevance for epithelial tumor cell biology, where loss of cell-cell contacts has been implicated in regulating cell survival and motility.