Recent interest in the use of human hair keratins as a biomaterial has grown, fuelled by improvements in keratin extraction methods and better understanding of keratin bioactivity. The use of keratins as a bioactive coating for in vitro cell culture studies is an attractive proposition. In this light, the surface adsorption of human hair keratins onto tissue culture polystyrene surfaces has been investigated. Keratin density, nano-topography and hydrophobicity of keratin coated surfaces were characterized. To understand the cellular influence of these coated surfaces, murine L929 fibroblasts were cultured on them and evaluated for cytotoxicity, proliferation, metabolic activity and detachment behaviors compared to collagen type 1 coated surfaces. Keratins were deposited up to a density of 650 ng/cm2 when a coating concentration of 80 μg/ml or higher was used. The surface features formed by adsorbed keratins also changed in a coating concentration dependent manner. These surfaces improved L929 mouse fibroblast adhesion and proliferation in comparison to uncoated and collagen type 1 coated tissue culture polystyrene. Furthermore, the expression of fibronectin was accelerated on surfaces coated with solutions of higher keratin concentrations. These results suggest that human hair keratins can be used as a viable surface coating material to enhance substrate compliance for culturing cells.