Osteoporosis is a bone disease affecting more than 2 million people comprising 1 in 3 women and 1 in 5 men in Canada. One possible approach to prevent this disease is to stimulate the activity of osteoblasts (bone-forming cells) using food protein-derived bioactive peptides. In our previous study, an ACE inhibitory tripeptide LRW (Leu-Arg-Trp) was identified from pea protein. This work aims to investigate the effect of tripeptide LRW on promoting osteoblastic activity. The tripeptide LRW treatment (50 μM) in MC3T3-E1 cells increased cell proliferation (4-fold increase) as indicated by BrdU incorporation assay. Moreover, we found that tripeptide LRW stimulated osteoblastic differentiation by increasing the levels of type 1 collagen (COL1A2; 3-fold increase), alkaline phosphatase (ALP; 4-fold increase), and runt-related transcription factor 2 (Runx2; 2-fold increase) and the activation of the protein kinase B (Akt) signaling pathway. Furthermore, tripeptide LRW increased matrix mineralization as evidenced by Alizarin-S red staining and nodule formation, osteoprotegerin levels (OPG; 2-fold increase), and wound healing based on cell migration assay. Overall, pea protein-derived bioactive peptide LRW can positively modulate the activity of osteoblasts probably via the Akt/Runx2 pathway, indicating its potential use for the prevention of osteoporosis.