As an emerging nanomaterial, graphene quantum dots (GQDs) have shown enormous potential in theranostic applications. However, many aspects of the biological properties of GQDs require further clarification. In the present work, we prepared two sizes of GQDs and for the first time investigated their membrane permeabilities, one of the key factors of all biomedical applications, and transport mechanisms on a Madin Darby Canine Kidney (MDCK) cell monolayer. The experimental results revealed that under ∼300 mg L(-1), GQDs were innoxious to MDCK and did not affect the morphology and integrity of the cell monolayer. The Papp values were determined to be 1-3 × 10(-6) cm s(-1) for the 12 nm GQDs and 0.5-1.5 × 10(-5) cm s(-1) for the 3 nm GQDs, indicating that the 3 nm GQDs are well-transported species while the 12 nm GQDs have a moderate membrane permeability. The transport and uptake of GQDs by MDCK cells were both time and concentration-dependent. Moreover, the incubation of cells with GQDs enhanced the formation of lipid rafts, while inhibition of lipid rafts with methyl-β-cyclodextrin almost eliminated the membrane transport of GQDs. Overall, the experimental results suggested that GQDs cross the MDCK cell monolayer mainly through a lipid raft-mediated transcytosis. The present work has indicated that GQDs are a novel, low-toxic, highly-efficient general carrier for drugs and/or diagnostic agents in biomedical applications.