Silk-based materials have garnered attention for use as medical supplies due to their mechanical toughness and low cytotoxicity. Silkworm silk has been applied as surgical sutures for decades. In contrast, the utilization of spider silk is limited mainly because of its scarcity. Although the biomimicry of spider silk has been developed using recombinant protein expression systems with the use of genetic engineering, the product often results in lower molecular weight and a lack of the N- or C-terminal regions. The incomplete sequence of the spider silk-like protein prevents the objective evaluation of the native spider silk as a medical application and retards the development of spider silk-inspired materials. Here, we reeled the native spider silk directly from live spiders and investigated the cell adhesion behavior based on three kinds of surface topography of spider silk-based substrates, namely, fibers, films, and non-woven fabrics. The cell adhesion behavior was largely influenced by the surface micro/nanostructure rather than the wettability of the surface. This study will contribute to promote the utilization of spider silk in the medical field as a candidate for promising bio-based fibers in the context of sustainable development goals.