As a new biomaterial, recombinant spider silk protein has attracted much attention in tissue engineering. The pNSR-16/ BL21(DE3)pLysS strains fermented and produced the recombinant spider silk protein, which was then cast into scaffolds. NIH-3T3 cells were cultivated with extractions of the scaffolds in vitro. The cytotoxicity of scaffolds was analyzed with a MTT assay. The performances of cells adhesion, growth and expression on the scaffolds were observed with SEM, HE staining and immunohistochemistry. Compared with the control, the extract fluid of materials culturing the NIH-3T3 cells was not apparently different. NIH-3T3 cells could adhere and grow on the scaffolds and secret FGF-2. The pNSR-16 recombinant spider silk protein scaffolds has satisfactory cytocompatibility and the scaffolds are ideal scaffold material for tissue engineering.