Mesenchymal stem cells (MSCs) have exciting medical potential, especially those that are genetically modified. Non-viral methods for genetic modification present several advantages over viral methods, and hence, such methods have been optimized for MSCs from a variety of species. Although equine MSCs have great importance, prior to this study, effective non-viral transfection parameters for these cells had not been determined. Here both chemical and physical transfection methods were optimized for equine MSCs, using red fluorescent protein as a reporter gene. Chemical reagents were optimized for reagent-to-DNA ratio, transfection solution plating volume, and cell density. Additionally, ideal voltage, cell concentration, DNA concentration, buffer, and pulse number settings were discovered for electroporation. The method resulting in highest gene expression was electroporation with one 30-msec pulse at 170 volts in Opti-Mem buffer. This technique resulted in transfection of 54% of the cell population, a percentage that is on the higher end of nonviral MSC transfection efficiencies. The transfection parameters determined in this study will undoubtedly be useful to all researchers who wish to genetically modify equine MSCs.