Cellular therapy using adipose tissue-derived mesenchymal stromal cells (at-MSCs) has garnered attention for the treatment of bone defects. Therefore, preconditioning strategies to enhance the osteogenic potential of at-MSCs could optimize cell therapy outcomes, and photobiomodulation (PBM) therapy has emerged as an effective, noninvasive, and low-cost alternative. This study explored the impacts of PBM on at-MSCs differentiation and the subsequent repair of bone defects treated with cell injection. Rat at-MSCs were cultured and irradiated (at-MSCs) following the PBM protocol (660 nm; 20 mW; 0.714 W/cm; 0.14 J; 5 J/cm). Cellular differentiation was assessed based on the expression of gene and protein markers. Reactive oxygen species (ROS) were detected using fluorescence. At-MSCs were injected into 5-mm calvarial lesions, and bone formation was analyzed using micro-CT and histological evaluations. At-MSCs were used as control. Data were analyzed using the ANOVA or t-test. At-MSCs exhibited high levels of gene and protein runt-related transcription factor-2 (Runx2) and alkaline phosphatase (Alp) expression. PBM increased ALP activity and significantly reduced ROS levels. In addition, PBM increased the expression of Wnt pathway-associated genes. In vivo, there was an increase in the morphometric parameters, including bone volume, percentage of bone volume, bone surface area, and trabecular number, in at-MSCs-treated defects compared with those in the control. These findings suggest that PBM enhances the osteogenic potential of at-MSCs, thereby supporting the advancement of improved cellular therapies for bone regeneration.