In this study, we evaluated the osseoconductivity of Type 316L stainless steel with self-organized nanopores of three different average diameters (26, 90, and 177 nm), formed by anodic polarization. The proliferation, alkaline phosphatase activity, and morphology of MC3T3-E1 mouse osteoblast-like cells, cultured on the self-organized nanopores were evaluated. The cell densities on samples with the nanopores were higher than those on mechanically nished surfaces that were mirror-polished or ground with #2000 SiC paper. In particular, the highest cell density and alkaline phosphatase activity were obtained on the nanoporous sample with the smallest diameter of 26 nm. Cells on the samples with 26 nm nanopores extended further and spread more lopodia compared with cells on samples with the other surface morphologies. Therefore, we concluded that self-organized nanopores with an optimal diameter (e.g., 26 nm) on Type 316L stainless steel could enhance long-term cell activity.