Tissue engineering with a combination of stem cells and nanofibrous scaffolds has attracted interest with regard to bone regeneration applications. In the present study, human induced pluripotent stem cells (iPSCs) were cultured on polymeric nanofibrous polyethersulfone (PES) with and without plasma treatment. The capacity of PES and plasma-treated PES (Plasma-PES) scaffolds to support the proliferation and osteogenic differentiation of iPSCs was investigated by MTT assay and for common osteogenic markers such as alkaline phosphatase activity, calcium mineral deposition and bone-related genes. Plasma-PES scaffolds with or without iPSCs were subsequently used to evaluate bone regeneration of critical-size defects in the rat by digital mammography, multislice spiral-computed tomography imaging and histological analysis. The results of in vitro analysis showed that plasma treatment significantly enhanced iPSC proliferation and osteogenesis. After 8 weeks of iPSC-loaded Plasma-PES implantation, no mortality or complication was observed in animals or at the site of surgery. Imaging analysis revealed more extensive bone reconstruction in rats receiving nanofibers compared with untreated control groups. Moreover, Plasma-PES seeded with iPSCs induced the highest regeneration of bone defects among all groups. These findings were confirmed by histological staining. Affective osseointegration was observed in implanted scaffolds. Thus, plasma-treated nanofibrous scaffolds are suitable tissue-engineered matrices for supporting the proliferation and osteogenic differentiation of iPSCs and might also be appropriate for the reconstruction of bone defects.