Gene-directed tissue repair offers the clinician, human or veterinary, the chance to enhance cartilage regeneration and repair at a molecular level. Non-viral plasmid vectors have key biosafety advantages over viral vector systems for regenerative therapies due to their episomal integration however, conventional non-viral vectors can suffer from low transfection efficiency. Our objective was to identify and validate in vitro a novel non-viral gene expression vector that could be utilized for ex vivo and in vivo delivery to stromal-derived mesenchymal stem cells (MSCs). Minicircle plasmid DNA vector containing green fluorescent protein (GFP) was generated and transfected into adipose-derived MSCs from three species: canine, equine and rodent and transfection efficiency was determined. Both canine and rat cells showed transfection efficiencies of approximately 40% using minicircle vectors with equine cells exhibiting lower transfection efficiency. A Sox9-expressing minicircle vector was generated and transfected into canine MSCs. Successful transfection of the minicircle-Sox9 vector was confirmed in canine cells by Sox9 immunostaining. This study demonstrate the application and efficacy of a novel non-viral expression vector in canine and equine MSCs. Minicircle vectors have potential use in gene-directed regenerative therapies in non-rodent animal models for treatment of cartilage injury and repair.