miR-20a-5p has recently been identified to induce adipogenesis of established adipogenic cell lines in our previous study. However, its role and molecular mechanisms in the regulation of adipocyte lineage commitment of bone marrow-derived stromal cells (BMSCs) still need to be explored. In this report, we demonstrated the expression of miR-20a-5p was promoted gradually during adipogenic differentiation in BMSCs. We also confirmed that miR-20a-5p has a positive function in the adipogenic differentiation of BMSCs by gain-of-function study with overexpression lentivirus or synthetic mimics of miR-20a-5p, and loss-of-function study with sponge lentivirus or synthetic inhibitor of miR-20a-5p. Dual luciferase reporter assay, GFP repression assay and Western blotting suggested Kruppel-like factor 3 (Klf3) was a direct target of miR-20a-5p. Furthermore, siRNA-mediated silencing ofKlf3recapitulated the potentiation of adipogenesis induced by miR-20a-5p overexpression, whereas enhanced expression ofKlf3attenuated the effect of miR-20a-5p. AsKlf3was reported to play an inhibitory role in adipogenesis at the initial stage of differentiation, the findings we present here indicate that miR-20a-5p promotes adipocyte differentiation from BMSCs by targeting and negatively regulatingKlf3in the early phase during the procedure of adipogenesis.