We demonstrate that the pore size of a nanoporous anodic aluminum oxide (AAO) membrane is critical for cell viability and cell adhesion upon HeLa cell growth on it, while other properties of membranes such as surface wettability and pore fraction show no such effects. After a human cancer cell, HeLa cell line, was seeded on AAO membranes with various pore sizes ranging from 18 to 150 nm, the adhered cell population and 3‐(4,5‐dimethyltiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay were measured. The cell adhesion and viability on the membrane with small pores (18 nm) were similar to those on the nonporous reference; however, they gradually decreased as the pore size increased. The decreased viability involves cell death as well as poor cell adhesion. We found that cyclo(l‐arginylglycyl‐l‐α‐aspartyl‐d‐phenylalanyl‐l‐cysteinyl), a well‐known integrin receptor antagonist, drastically decreased the cell adhesion to the membranes. The data indicate that the cell adhesion to the nanoporous membranes may be mediated by integrin on the cell surface, plausibly explaining the pore size dependence of the cell adhesion and viability. Implications of the present results to related fields such as implants are discussed.