Osteoporosis, the most prevalent metabolic bone disorder, is a major public health issue. Previous studies indicated the potential of food components in mitigating the risks of osteoporosis. The study aimed to evaluate the potential of canola protein hydrolysates (CPH) on osteoclastogenesis using a pre-osteoblast cell MC3T3-E1. Twenty-two CPHs were prepared by 12 different proteases, either individually or in combination. Three CPHs, prepared by trypsin (CPH-T), Protex 6L (CPH-P), and the combination of Protex 6L and thermoase (CPH-PT) showed promising activity in promoting in vitro bone formation. CPH-T and CPH-PT improved cell proliferation at a concentration of 10 ug/ml, while all three hydrolysates exhibited cytotoxicity at 1000 ug/ml. All three hydrolysates promoted the level of runt-related transcription factor 2 (RUNX2) and type I collagen, and mineralization in osteoblast cells, in a dose-dependent manner. Additionally, these three hydrolysates elevated the osteoprotegerin (OPG) level and reduced the level of receptor activator of nuclear factor kappa-B ligand (RANKL). This study indicated the activity of CPHs in the promotion of bone formation and prevention of osteoclastogenesis, suggesting the potential of CPHs as a promising functional food ingredient against osteoporosis.