Nitrocellulose membranes, one of the most important and oldest cellulose derivatives, are commonly used for nucleic acid and protein detection in research and diagnostic applications. However, a limited number of studies have explored whether they can act as scaffolds for cell growth. In this study, we investigated this polymeric material for its ability to support the growth of human cells. Eight established cell lines were examined for adherence, growth, spread, and survival on nitrocellulose membranes by optical microscopy after hematoxylin and eosin and/or immunocytochemical staining and by scanning electron microscopy. Apoptosis and leakage of lactate dehydrogenase (LDH) were also assessed. All cells readily adhered to and spread on the surface of nitrocellulose membranes as well as coverslips, and the cells maintained the expression of digestive system-specific genes. No significant change was detected in apoptosis or leakage of LDH from cells grown on nitrocellulose membranes. These results suggested that nitrocellulose membranes have a suitable cytocompatibility towards human cells and that they might be used for tissue-engineering scaffolds. Moreover, we demonstrate an additional and underused property of nitrocellulose of specific relevance to microscopic imaging, as it can be rendered virtually transparent, thus the cells growing on such membranes can be observed directly under an optical microscope after staining.