Chronic exposure to commercial glucose-based peritoneal dialysis fluids during peritoneal dialysis induces peritoneal membrane damage leading to ultrafiltration failure. In this study the role of protein kinase C (PKC) α in peritoneal membrane damage was investigated in a mouse model of peritoneal dialysis. We used 2 different approaches: blockade of biological activity of PKCα by intraperitoneal application of the conventional PKC inhibitor Go6976 in C57BL/6 wild-type mice and PKCα-deficient mice on a 129/Sv genetic background. Daily administration of peritoneal dialysis fluid for 5 weeks induced peritoneal upregulation and activation of PKCα accompanied by epithelial-to-mesenchymal transition of peritoneal mesothelial cells, peritoneal membrane fibrosis, neoangiogenesis, and macrophage and T cell infiltration, paralleled by reduced ultrafiltration capacity. All pathological changes were prevented by PKCα blockade or deficiency. Moreover, treatment with Go6976 and PKCα deficiency resulted in strong reduction of proinflammatory, profibrotic, and proangiogenic mediators. In cell culture experiments, both treatment with Go6976 and PKCα deficiency prevented peritoneal dialysis fluid–induced release of MCP-1 from mouse peritoneal mesothelial cells and ameliorated transforming growth factor-β1–induced epithelial-to-mesenchymal transition and peritoneal dialysis fluid–induced MCP-1 release in human peritoneal mesothelial cells. Thus, PKCα plays a crucial role in the pathophysiology of peritoneal membrane dysfunction induced by peritoneal dialysis fluids, and we suggest that its therapeutic inhibition might be a valuable treatment option for peritoneal dialysis patients.