Multipotent mesenchymal stromal cells (MSCs) have gained tremendous attention as potential therapeutic agents for the treatment of orthopedic diseases. Promising results have been obtained after application of MSCs for treatment of tendon and joint disease in the equine model, making it appear favorable to use these results as a basis for the translational process of the therapy. However, while the horse is considered a highly suitable model for orthopedic diseases, knowledge is lacking regarding the level of analogy of equine MSCs and their human counterparts. Therefore, the aim of this study was to assess the properties of human and equine adipose- and tendon-derived MSCs in a direct comparison. Basic properties of human and equine MSCs from both tissues were similar. The cells expressed CD29, CD44, CD90, and CD105 and lacked expression of CD73, CD14, CD34, CD45, CD79α, and MCHII/HLA-DR. No significant differences were found between proliferation potential of human and equine MSCs in early passages, but recovery of nucleated cells after tissue digestion as well as proliferation in later passages was higher in equine samples (p < 0.01). All samples showed a good migration capacity and multilineage differentiation potential. However, while osteogenic differentiation was achieved in all equine samples, it was only evident in five out of nine human tendon-derived samples. Human MSCs further showed a higher expression of collagen IIIA1 and tenascin-C, but lower expression of decorin and scleraxis (p < 0.01). Although revealing some potentially relevant differences, the study demonstrates a high level of analogy between human and equine MSCs, providing a basis for translational research in the equine model according to the guidelines issued by the authorities.