Postmenopausal osteoporosis compromises bone regeneration and elevates the risk of fracture nonunion, underscoring the necessity for more effective regenerative strategies. In this study, we investigated the regenerative efficacy of a combined therapy consisting of photobiomodulation (PBM), preconditioned adipose-derived stem cells (ADSCs), and human decellularized and mineralized placental matrix scaffolds (hDMPMS), in an ovariectomized induced osteoporosis (OVX) rat with critical-sized femoral defects (CSFD). We surgically created critical-size femoral defects (CSFD) in the distal metaphysis of the left and right femurs of thirty OVX rats. After inducing CSFD, hDMPMSs were implanted into each defect site. The animals were then randomly divided into five groups (n = 6 each group): (1) Control, (2) ‎Scaffold, (3) Scaffold + Cell, (4) Scaffold + PBM (810 nm, 1.2 J/cm²), and ‎‎(5) Scaffold + Cell + PBM. Eight weeks after treatment, the defect sites in the left femoral bone were harvested and subjected to mechanical compression testing, bone density measurement, as well as gene expression and histological analyses. One-way ANOVA revealed significant (P < 0.001) improvements in biomechanical strength, histological parameters, and bone density in all treatment groups compared to the control group. Among them, the combination treatment group (Cell + PBM) demonstrated the greatest enhancement across all evaluated parameters. Gene expression analysis revealed elevated levels of miR-21 and RUNX2 in all treatment groups, with the highest expression observed in the combination treatment group compared to the control group. The data indicate a substantial enhancement in bone repair for osteoporotic CSFDs treated with the combined therapy of hDMPMSs, PBM-preconditioned ADSCs, and PBM. This effect is likely mediated, at least in part, through modulation of bone remodeling processes and activation of the miR-21/RUNX2 osteogenic axis.