Prolonged endoplasmic reticulum (ER) stress contributes to cell apoptosis and interferes with bone homeostasis. Although photobiomodulation (PBM) might be used for ER stress‐induced diseases, the role of PBM in relieving cell apoptosis remains unknown. During ER stress, glycogen synthase kinase‐3β (GSK‐3β) is critical; however, its functions in PBM remain uncertain. Thus, this study aimed to investigate the role of GSK‐3β in 625 nm light‐emitting diode irradiation (LEDI) relieving tunicamycin (TM)‐induced apoptosis. Based on the results, pre‐625 nm LEDI (Pre‐IR) phosphorylated GSK‐3β via ROS production. Compared with the TM group, Pre‐IR + TM group reduced the phosphorylation of the α‐subunit of eukaryotic translation initiation factor 2 (eIF‐2α) and B‐cell lymphoma protein 2 (Bcl‐2)‐associated X (Bax)/Bcl‐2 ratio through regulating GSK‐3β. Furthermore, a similar tendency was observed between Pre‐IR + TM and Pre‐LiCl+TM groups in preventing TM‐induced early and late apoptosis. In summary, this study suggests that the Pre‐IR treatment in TM‐induced ER stress is beneficial for preventing cell apoptosis via GSK‐3β phosphorylation.