Graphene derivatives are known to be suitable for biomedical applications, especially for stem cell-based therapies. Herein, we report the size effects of graphene oxide (GO) on differentiation of human adipose-derived mesenchymal stem cells (hADMSCs), using micro-sized (MGO) and nano-sized graphene oxide (NGO) sheets. The MGO and NGO sheets having lateral sizes of 1–10 μm and 100–300 nm, respectively, are coated on glass substrates by drop casting. The hADMSCs grown on the MGO-coated substrates show enhanced cell spreading and proliferation rate when compared with those of NGO counterpart, regardless of their densities. The GO size-dependency of hADMSCs becomes more prominent when it comes to their differentiation capabilities. After 4 weeks of differentiation under the same culture conditions, the osteogenesis of hADMSCs grown on the MGO-coated substrate is particularly higher than that on the NGO-coated substrate. The difference in osteogenic differentiation of hADMSCs is found to be most dominant after 21–28 days of differentiation according to the calcification level of osteoblasts. These finding signify the importance of graphene size in controlling the osteogenesis of hADMSCs, which may shed new insight into the use of graphene-based materials for stem cell research and therapy.