Multipotent mesenchymal stromal cells (MSC) have attracted interest for their potential in allogeneic cytotherapy, partly due to their ability to secrete immunosuppressive factors. However, development of efficacious and reproducible therapies is hampered by a knowledge-gap in equine MSC characterization. I hypothesized that equine MSC can be consistently isolated from cord blood, have unique and reproducible marker expression, which includes cytoplasmic Toll-like receptor (TLR) 3 and TLR4, and in vitro suppress lymphoproliferation. I aimed to establish the immunophenotype of cord blood-derived-(CB)-MSC before and after cryopreservation and confirm their ability to suppress in vitro lymphoproliferation. Cultured CB-MSC expressed CD29, CD44, CD90, and not MHC I, MHC II, CD4, CD8, CD11a/18, and CD73 before and after cryopreservation. CB-MSC suppressed in vitro lymphoproliferation and constitutively expressed TLR4. These findings suggest anti-inflammatory properties of CB-MSC. Lack of MHC I expression suggests reduced risk of allorejection. The relationship between TLR4 and lymphocyte function requires further investigation.