Self-assembling peptide (SAP) nanofiber hydrogel scaffolds have become increasingly important in tissue engineering due to their outstanding bioactivity and biodegradability. However, there is an initial concern on their long-term clinical use, since SAPs made of L-form amino acid sequences are sensitive to enzymatic degradation. In this study, we present a designer SAP, D-RADA16, made of all D-amino acid. We investigated the nanofiber morphology of D-RADA16, its potential for the culture of bone marrow-derived mesenchymal stem cells (BMSCs), and the proteolytic resistance of the biomaterial. The results revealed that D-RADA16 exhibited stable β-sheets and formed interwoven nanofiber scaffolds in water. D-RADA16 and L-RADA16 hydrogel scaffolds were both found to promote the proliferation and migration of rat BMSCs in the 3D cell culture microenvironment. Furthermore, the D-RADA16 scaffolds exhibited a higher proteolytic resistance against proteinase K than the L-RADA16 scaffolds. These observations indicate that D-RADA16 hydrogel scaffolds have excellent bioactivity, biocompatibility and biostability, and thus may serve as promising candidates for long-term application in vivo.