Thin cellulose nanofiber (CNF) nanostructured substrates with varying roughness, stiffness (Young's modulus), porosity, and swelling properties were produced by varying the conditions used during fabrication. It was shown that with increased heat exposure, CNF substrate porosity in an aqueous state decreased while Young's modulus in a water submerged state increased. In this study, the adhesion and viability of mesenchymal stem cells (MSCs) cultured on this CNF substrate will be presented. Viability of D1/BALBc MSCs were assessed for 24 and 48 h, and it was shown that depending on the CNF substrate the viability varied significantly. The adhesion of MSCs after 6 and 24 h was conditional on material mechanical properties and porosity of the CNF in cell culture conditions. These results suggest that material properties of CNF nanostructured substrate within the aqueous state can be easily tuned with curing step without any chemical modification to the CNF and that these changes can affect MSC viability in cell culture.