Protein kinase D (PKD) is a novel protein serine kinase which has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was: (i) to define the activation of PKD in intestinal epithelial cells treated with hydrogen peroxide (H₂O₂), an agent which induces oxidative stress, and (ii) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H₂O₂ in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective protein kinase C (PKC)- inhibitor, and small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK) specific inhibitor, Y27632, as well as C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H₂O₂-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H₂O₂-induced intestinal cell apoptosis was enhanced by PKD siRNA. Taken together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells, and this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications to intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants).