In this study, we evaluated the role of WNT16 in regulating bone size, an important determinant of bone strength. Mice with targeted disruption of the Wnt16 gene exhibited a 24% reduction in tibia cross-sectional area at 12 weeks of age compared with that of littermate wild-type (WT) mice. Histomorphometric studies revealed that the periosteal bone formation rate and mineral apposition rate were reduced (P < .05) by 55% and 32%, respectively, in Wnt16 knockout (KO) vs WT mice at 12 weeks of age. In contrast, the periosteal tartrate resistant acid phosphatase-labeled surface was increased by 20% in the KO mice. Because mechanical strain is an important physiological regulator of periosteal bone formation (BF), we determined whether mechanical loading-induced periosteal BF is compromised in Wnt16 KO mice. Application of 4800-μe strain to the right tibia using a 4-point bending loading method for 2 weeks (2-Hz frequency, 36 cycles per day, 6 days/wk) produced a significant increase in cross-sectional area (11% above that of the unloaded left tibia, P < .05, n = 6) in the WT but not in the KO mice (-0.2% change). Histomorphometric analyses revealed increases in the periosteal bone formation rate and mineral apposition rate in the loaded bones of WT but not KO mice. Wnt16 KO mice showed significant (20%-70%) reductions in the expression levels of markers of canonical (β-catenin and Axin2) but not noncanonical (Nfatc1 and Tnnt2) WNT signaling in the periosteum at 5 weeks of age. Our findings suggest that WNT16 acting via canonical WNT signaling regulates mechanical strain-induced periosteal BF and bone size.