Therapies using human mesenchymal stem cells (MSCs) combined with three-dimensional (3D) printed scaffolds are a promising strategy for bone grafting. But the harvest of MSCs still remains invasive for patients. Human synovial fluid MSCs (hSF-MSCs), which can be obtained by a minimally invasive needle-aspiration procedure, have been used for cartilage repair. However, little is known of hSF-MSCs in bone regeneration. Polyetherketoneketone (PEKK) is an attractive bone scaffold due to its mechanical properties comparable to bone. In this study, 3D-printed PEKK scaffolds were fabricated using laser sintering technique. hSF-MSCs were characterized and cultured on PEKK to evaluate their cell attachment, proliferation, and osteogenic potential. Rabbit calvarial critical-sized bone defects were created to test the bone regenerative effect of PEKK with hSF-MSCs. In vitro results showed that hSF-MSCs attached, proliferated, and were osteogenic on PEKK. In vivo results indicated that PEKK seeded with hSF-MSCs regenerated twice the amount of newly formed bone when compared to PEKK seeded with osteogenically-induced hSF-MSCs or PEKK scaffolds alone. These results suggested that there was no need to induce hSF-MSCs into osteoblasts prior to their transplantations in vivo. In conclusion, the combined use of PEKK and hSF-MSCs was effective in regenerating critical-sized bone defects.