Objective: Avocado/soybean unsaponifiables (ASU) is indicated as supplement for the palliative treatment of rheumatoid arthritis and osteoarthritis. In vitro studies have shown that ASU enhances Transforming Grown Factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) expression. The objective of this study was to evaluate the influence of ASU (Piascledine 300, Expanscience Lab, France) on bone healing in critical-size calvarial defects (CCD) in the presence and absence of bone substitutes. Method: A full thickness CCD defect (Ø 5mm) was made in each of 84 male wistar rats. The defects were implanted with either deproteinized bovine bone (DBB) (Bio-Oss, Geistlich Pharma, Switzerland) or a biphasic tricalcium phosphate/hydroxyapatite (TCP/HA) (Straumann Bone Ceramic, Straumann, Switzerland) particulate material, or filled only with coagulum (COA). Starting 15 days prior to surgery, ASU (0.6 gr. per Kg body weight) or saline (CTR) was administered daily by gavage until sacrifice 15 or 60 days post-op (7 animals per group at each observation period). Tissue composition within the defect was estimated by histomorphometry. Non-paired T- test, one-way Anova, and Tukey´s tests were used to evaluate differences (P < 0.05). Result: Bone fill in the COA-ASU group was significantly higher comparing to that in the COA-CTR group, both at 15- (46.4±10.4 vs. 29.0±8.8) and 60 days (52.1±6.1 vs. 42.7±5.2). No significant differences in bone fill were observed between DBB-ASU and DBB-CTR at day 15 (29.3±4.5 vs. 31.4±7.5) and day 60 (33.1±4.6 vs. 39.9±10.5), as well as between TCP/HA-ASU and TCP/HA-CTR at day 15 (33.3±6.7 vs. 33.0±6.5) and day 60 (31.7±5.0 vs. 39.6±8.7). Bone fill in COA-ASU group was significantly more than in DDB-ASU and TCP/HA-ASU groups at day 60. Conclusion: ASU enhanced bone healing in CCD originally filled only with coagulum, while implantation of DDB and TCP/HA appeared to obstruct the positive effect of ASU.