Rabbit cells and models are frequently used in pharmacological experiments and toxicity tests and are particularly useful for corneal transplant experiments. Here, we obtained a corneal endothelial cell line from normal rabbit corneal endothelium that had acquired infinite proliferative potential without requiring gene transfer. These infinitely proliferative rabbit corneal endothelial cells (iRCECs) could be cultured for >250 generations and exhibited a greater ability to proliferate than did normal rabbit corneal endothelial cells (RCECs) cultivated by conventional methods. The results of reverse transcriptase-polymerase chain reaction (RT-PCR) and immunostaining analyses revealed that the expression profiles of corneal endothelial markers, such as collagen type VIII1 and Na+/K+-ATPase, were similar to those of RCECs and in vivo corneal endothelium. Scanning electron microscopy showed that many microvilli were present on the surface of the cells and that the ultrastructure was maintained. In addition, we verified that the iRCECs had similar levels of pump function as did RCECs using an Ussing chamber system. The results of a soft agar colony-formation assay suggested that the iRCECs were not tumourigenic. Taken together, our results demonstrated that the iRCECs exhibited gene expression profiles and properties that were equivalent to those of native rabbit corneal endothelium, making these cells useful for corneal endothelial research studies. Copyright (c) 2015 John Wiley & Sons, Ltd.