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Graphene has been proven to have great potential in medical applications. The goal of this study is to evaluate the toxicity of graphene on human microvascular endothelial cells (HMEC-1) under flowing conditions mimicking blood circulation. Graphene was prepared by the electrochemical exfoliation method and conjugated with poly(sodium-4-styrenesulfonate) (PSS) to improve its hydrophilicity and dispersion behavior. Under static cell growth conditions, 80 mg ml−1 of graphene reduced cell viability to 60% of the control. Graphene was observed to aggregate on the cell surface by interacting with filopodia. In contrast, graphene applied to cells under flowing conditions showed no significant negative influence on the cells regardless of graphene concentration, incubation time, and the exerted shear force. Graphene also showed lower toxicity towards cells in a flowing culture than multi-walled carbon nanotubes (MWCNTs) and carbon black. Together, these results suggest that the prolonged interaction of graphene with the cell surface is critical in causing cell damage. The lower circulatory toxicity of graphene compared to MWCNTs and carbon black also suggests that graphene is more suitable for use in drug delivery systems.