We have introduced well-defined nanopillar arrays of polyethylene glycol (PEG) as a platform for studying the adhesion and growth of cultured cardiomyocytes. The nanopillar arrays were fabricated by using a simple molding technique involving the placement of a patterned polyurethane acrylate mold on top of a drop-dispensed ultraviolet (UV) curable PEG polymer followed by UV exposure and mold removal. The adhesion and growth of cardiomyocytes turned out be guided by an external nanotopography, which has been characterized in terms of cell morphology and cytoskeletal arrangement. In particular, the nanopillars provided guiding posts to both elongating filopodia and expanding lamellipodia. Interestingly, the 3D growth of cardiomyocytes was mediated by the increased hydrophobicity of the nanostructured PEG substrate, indicating that the cell adhesion and growth is very sensitive to the nanotopography. The precise nanostructures of PEG-based polymer with controlled geometrical features presented in this study not only open opportunities for understanding and tailoring cell adhesion and growth, but could serve as a template for better tissue engineering by controlling cellular activities at the molecular level.