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We have recently demonstrated that heterologous transplantation of horse amniotic membrane-derived mesenchymal cells (AMCs) can be useful for cell therapy applications in tendon diseases, and hypothesized that these cells may promote tendon repair via paracrine-acting molecules targeting inflammatory processes. To test this hypothesis, here we examined the immunomodulatory characteristics of AMCs and of their conditioned medium (AMC-CM) in vitro, and studied the potential therapeutic effect of AMC-CM in thirteen different spontaneous horse tendon and ligament injuries in vivo. Our results demonstrate that AMCs are capable of inhibiting peripheral blood mononuclear cell (PBMC) proliferation after allogenic stimulation either when cocultured in cell-to-cell contact, or when the two cell types are physically separated by a transwell membrane, suggesting that soluble factors are implicated in this phenomenon. Our hypothesis is further supported by the demonstration that PBMC proliferation is inhibited by AMC-CM. In our in vivo studies, no significant adverse effects were observed in treated tendons, and clinical and ultrasonographical evaluation did not reveal evidence of inappropriate tissue or tumor formation. Clinical outcomes were favorable and the significantly lower rate (15.38%) of reinjuries observed compared to untreated animals, suggests that treatment with AMC-CM is very efficacious. In conclusion, this study identifies AMC-CM as a novel therapeutic biological cell-free product for treating horse tendon and ligament diseases