Increased fibronectin expression is a key feature of diabetic angiopathy. We have previously shown that nuclear factor-κB (NF-κB) mediates fibronectin expression in endothelial cells and in organs affected by diabetes complications. p300, a transcription coactivator, may regulate NF-κB activity via poly(ADP-ribose) polymerase (PARP) activation. Hence, we examined the role of p300 in fibronectin expression in diabetes. High glucose induced fibronectin expression in the endothelial cells, which was associated with increased p300, PARP activity, and NF-κB activation. This p300 alteration is mediated by mitogen-activated protein kinase and protein kinase C and B. We then used p300 small interfering RNA (siRNA) and showed decreased fibronectin and PARP expression, as well as NF-κB activation, in the endothelial cells. Examination of the heart tissues of streptozotocin-induced diabetic mice revealed increased fibronectin and p300 mRNA. Intravenous injection of p300 siRNA resulted in decreased p300 levels and normalized fibronectin expression in the heart. We further investigated retinal tissues from streptozotocin-induced diabetic rats treated with intravitreal p300 siRNA injection. Similar to the heart, p300 siRNA inhibited fibronectin expression in the retina of the diabetic animals. These results indicate that transcriptional coactivator p300 may regulate fibronectin expression via PARP and NF-κB activation in diabetes.