Objective To investigate whether bone marrow mesenchymal stem cells （BMSCs） can promote periosteal distraction osteogenesis （PDO）. Methods BMSCs isolated from New Zealand white rabbits were cultured by the whole bone marrow adherent method and induced to differentiate into osteocytes, adipocytes and chondrocytes in vitro. Poly l-lactic acid （PLLA） scaffold produced by three-dimensional printing （3D printing） technology was used to distract the calvarial periosteum of rabbit. At the end of distraction period, BMSCs were injected into the gap between periosteum and skull in experimental group while the control group received equal normal saline （NS）. The distraction parts of rabbits＂ skull were collected after 4 and 8 weeks of consolidation periods. The newly formed bone of both groups was evaluated by Micro-CT scanning and histological staining. Results BMSCs obtained by the whole bone marrow culture method had the ability of multi-directional differentiation. Micro-CT scanning showed that there were new tissues formed in the distraction area at each time point both in experimental group and control group. The bone volume （BV）, bone volume/tissue volume （BV/TV）, bone mineral density （BMD）, trabecular number （Tb.N） and trabecular thickness （Tb.Th） in experimental group were higher than that in control group, however, the trabecular separation （Tb.Sp） in the experimental group was lower than that of the control group. HE staining also indicated new bone formation both in experimental group and control group. Compared with the control group, the newly formed trabecular bone in the experimental group was thicker and maturer, and the boundary between the new bone and the skull cortex was not obvious. Conclusion Injection of autologous BMSCs into the PDO region of rabbit skull can supplement the deficiency of osteocytes, which is beneficial to PDO.