Objective: Lenalidomide (LEN) has emerged as a promising therapeutic option for the management of various hematologic malignancies. Although its direct mechanisms of action on malignant cells have been studied intensively, its effects on the stromal compartment of bone marrow have not yet been analyzed systematically. Therefore, we investigated whether LEN alters the functional capacity of mesenchymal stromal cells (MSCs) as the main cellular component of the bone marrow microenvironment. In addition to their growth and differentiation characteristics, we focused on the ability of MSC to modulate T-cell function and support hematopoietic stem cells (HSCs). Materials and Methods: Bone marrow-derived MSCs were exposed to LEN (10 μM), and differences in proliferation, phenotype, inhibition of T-cell proliferation, and differentiation capacity were analyzed. A Boyden chamber assay was used to test the migratory potential of HSC toward the conditioned medium of LEN-treated or untreated MSCs, and the stromal cell-derived factor-1 (SDF-1) concentrations in these supernatants were determined by enzyme-linked immunosorbent assay. Results: Treatment of MSCs with LEN did not affect their growth rate, proliferation, osteogenic and adipogenic differentiation potential, or capacity to inhibit T-cell proliferation. However, LEN treatment increased the average of mean fluorescence intensity of CD29 and CD73 by 15 and 22%, respectively. Interestingly, LEN reduced SDF-1 by MSCs by 32% compared to that of control cells. As a functional consequence, the serum-free supernatant of LEN-treated MSCs had a significantly lower potential to induce the directed migration of CD34⁺ HSCs. Conclusion: LEN can modulate the expression of cell surface molecules and the chemokine secretion of MSCs in vitro. These effects might contribute to the clinical effects of the compound in vivo for patients with hematological malignancies.