Posterolateral lumbar fusion (PLF) is a commonly performed surgical procedure for the treatment of pathological conditions of the lumbosacral spine. In the present study, we evaluated an autologous bone graft substitute (ABGS) containing rhBMP6 in autologous blood coagulum (ABC) and synthetic ceramics used as compression resistant matrix (CRM) in the rabbit PLF model. In the pilot PLF rabbit experiment, we tested four different CRMs (BCP 500–1700 μm, BCP 1700–2500 μm and two different TCP in the form of slabs) which were selected based on achieving uniform ABC distribution. Next, ABGS implants composed of 2.5 mL ABC with 0.5 g ceramic particles (TCP or BCP (TCP/HA 80/20) of particle size 500–1700 μm) and 125 μg rhBMP6 (added to blood or lyophilized on ceramics) were placed bilaterally between transverse processes of the lumbar vertebrae (L5-L6) following exposition and decortication in 12 New Zealand White Rabbits observed for 7 weeks following surgery. Spinal fusion outcome was analysed by μCT, palpatory segmental mobility testing and selected specimens were either tested biomechanically (three-point bending test) and/or processed histologically. The total fusion success rate was 90.9% by both μCT analyses and by palpatory segmental mobility testing. The volume of newly formed bone between experimental groups with TCP or BCP ceramics and the different method of rhBMP6 application was comparable. The newly formed bone and ceramic particles integrated with the transverse processes on histological sections resulting in superior biomechanical properties. The results were retrospectively found superior to allograft devitalized mineralized bone as a CRM as reported previously in rabbit PLF. Overall, this novel ABGS containing rhBMP6, ABC and the specific 500–1700 μm synthetic ceramic particles supported new bone formation for the first time and successfully promoted posterolateral lumbar fusion in rabbits. •A new ABGS containing rhBMP6 in autologous blood coagulum with synthetic ceramics (500-1700 μm) was tested in PLF rabbit model.•The fusion success rate was 90.9% by microCT analysis, palpatory segmental mobility and biomechanical testing.•This novel ABGS for the first time supports new bone formation and promotes PLF in rabbits.