Background: Emerging evidence suggests that secretome of mesenchymal stem cells has many anti-inflammatory and regenerative properties, which makes it a suitable candidate for the treatment of autoimmune and degenerative diseases. Dipeptidyl Peptidase-IV (DPP-IV)/CD26 and Aminopeptidase N (APN)/CD13 are ubiquitous ecto-enzymes which can digest various substrates including some chemokines and neuropeptides that are involved in inflammatory conditions. Objective: To evaluate the enzymatic activity of DPP-IV/CD26 and APN/CD13 in MSC conditioned media (MSC-CM). Methods: The MSCs were isolated from the mouse’s abdominal adipose tissues and were cultured without or with preconditioning by adding 2 µg/mL phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). The levels of interleukin-10 (IL-10), nitric oxide (NO), as well as the enzymatic activities of DPP-IV/CD26 and APN/CD13 were measured in MSC-CM. Results: The level of IL-10 and the enzyme activity of APN/CD13 did not show any changes in the MSC-CM of stimulated and non-stimulated cells. However, NO production was increased after treatment by LPS or PMA; nevertheless, the DPP-IV/CD26 activity was decreased in MSC-CM merely following the stimulation of cells with LPS. Conclusion: Our results indicated that MSC‐secretome had DPP-IV/CD26 and APN/CD13 activity. The DPP-IV/CD26 activity was decreased following stimulation of MSCs by toll-like receptor 4 agonist. Further studies are needed to reveal the possible contribution of DPP-IV/CD26 and APN/CD13 in the anti-inflammatory functions of MSC-CM.