The aim of the present work was to isolate, cultivate, differentiate, and conduct cellular characterization of mesenchymal stem cells (MSCs) derived from equine adipose tissue (eAT) and bone marrow (eBM). Isolated and characterized cells were used in racehorses suffering from a superficial flexor tendon injury. Equine adipose tissue collection was performed at the base of the horse tail, whereas eBM was aspirated from iliac crest. Mononuclear cell fraction was isolated and cultured. In vitro differentiation and molecular characterization at P3 of culture were performed. No statistically significant differences in the number of cell doublings were found among different culture passages (P > .05). Doubling time was greater for eBM than eAT (3.2 ± 1.5 vs. 1.3 ± 0.7; P < .05). Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian Blue and Oil Red O staining illustrated chondrogenesis and adipogenesis, respectively. Isolated cells resulted positive for CD90, CD44, and CD105, whereas negative for hematopoietic markers, CD14, CD45, and CD34. Using isolated cells for injured tendon therapy, no adverse reactions were observed, and all inoculated horses returned to race competitions. In vitro results revealed the immunophenotypic characterization of isolated cells similar to that observed in human MSCs from the same sources; furthermore, in the present study, their clinical use proves the safety of eBM-derived and eAT-derived MSCs and a successful outcome for the treated animals that returned to their previous level of sport activity.