The present study investigated the effects of egg yolk-derived peptide (YPEP) on osteogenic activities and MAPK-regulation of osteogenic gene expressions. The effects of YPEP on cell proliferation, alkaline phosphatase activity, collagen synthesis, and mineralization were measured in human osteoblastic MG-63 cells. Activation of MAPKs and downstream transcription factors such as extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2), p38, ELK1, and cJUN were examined using western blot analysis. YPEP dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. YPEP activated ERK1/2, p38, and ELK1 phosphorylation whereas JNK and cJUN were not affected by YPEP. The COL1A1 (collagen, type I, alpha 1), ALPL (alkaline phosphatase), and SPP1 (secreted phosphoprotein 1, osteopontin) gene expressions were increased while BGLAP (osteocalcin) was not affected by YPEP. The ERK1/2 inhibitor (PD98509) blocked the YPEP-induced COL1A1 and ALPL gene expressions as well as ELK1 phosphorylation. The p38 inhibitor (SB203580) blocked YPEP-induced COL1A1 and ALPL gene expressions. SPP1 gene expression was not affected by these MAPK inhibitors. In conclusion, YPEP treatment stimulates the osteogenic differentiation via the MAPK/ELK1 signaling pathway. These results could provide a mechanistic explanation for the bone-strengthening effects of YPEP.