Bone defect architecture has become an issue to resemble in scaffold properties, beside enhancing osteoinduction. Thus, a non-invasive injectable approach is proposed by using silk fibroin (SF)-based hydrogel containing magnetic nanohydroxyapatite (MHAp) and osteogenic growth peptide (OGP). This study aimed to disseminate the osteogenic differentiation potential of the hydrogel and its mechanism on canine periodontal ligament stem cells (cPDLSCs). The addition of MHAp and/or OGP into the hydrogel shows remarkable osteogenic differentiation by the elevated ALP activity, osteogenic marker expression, and biomineralization. Proteomics analysis results revealed that TGF-β, BMP, and Notch signaling pathways are involved in the injectable hydrogel-induced osteogenic differentiation of cPDLSCs, validated by inhibition assay. Overall, HG/OGP/MHAp shows potential to be used in clinical practice for treating alveolar bone defects.