This study investigated the impact of 808 nm laser photobiomodulation (PBM) on mitochondrial respiration and osteogenic protein expression (OCN, OPN, ALP, RUNX2, COL-1, BMP-2) in human dental pulp stem cells (hDPSCs) within a 3D hydrogel model. hDPSCs were isolated from third molars and maintained under hypoxic conditions. Cells received PBM at 5 and 15 J/cm2 using an 808 nm diode laser. The study showed that 808 nm PBM can alter mitochondrial respiration, with 5 J/cm2 enhancing osteogenic protein expression (OCN, ALP, OPN, RUNX2) but failing to sustain BMP-2 at 24 h. In contrast, 15 J/cm2 induced stronger upregulation and prolonged BMP-2 expression, suggesting an optimal dose for sustained osteogenic activity. BMP-2 was later downregulated, and COL-1 remained unchanged post-PBM. Importantly, this study indicates the dose-specific PBM modulation of mitochondrial respiration and protein expression, but further research is required to optimize treatment protocols.