The aim of the present study was to evaluate the cytotoxicity of pristine graphene monolayer and its utility as a scaffold for murine fibroblast L929 cell line. Cell viability, morphology, cytoskeleton architecture (microfilaments and microtubules), cell adhesion and migration into the scratch-wound area were determined using pristine graphene-coated microscopic slides. We found that fibroblasts cultured on pristine graphene monolayer exhibited changes in cell attachment, motility and cytoskeleton organization. Graphene was found to have no cytotoxicity on L929 fibroblasts and increased cell adhesion and proliferation within 24 h of culture. The area of cells growing on graphene was comparable to the area of fibroblasts cultured on glass. Migration of cells on the surface of graphene substrate appeared to be more regular in comparison to uncoated glass surface, however in both control (glass) and experimental (graphene) groups the scratch wound was closed after 48 h of culture. Taken together, our results indicate that pristine graphene monolayer is non-toxic for murine subcutaneous connective tissue fibroblasts and could be beneficial for recovery of damaged tissues after injury. These studies could be helpful in evaluating biocompatibility of graphene, which still remains ambiguous.