Autophagy is activated under radiation stress, which serves an important role in maintaining bone homeostasis. However, the underlying mechanisms of irradiation‑induced autophagy in bone homeostasis is not well understood. The present study aimed to determine the effects of radiation‑activated autophagy on pre‑osteoblastic MC3T3‑E1 cells. X‑ray irradiation activated autophagy in a dose‑dependent manner, with an increased fluorescence intensity of monodansylcadaverine staining, increased ratio of microtubule‑associated protein 1 light chain 3β (LC3)‑II/LC3‑I, decreased p62 expression, and increased ATG5 and beclin‑1 expression levels in MC3T3‑E1 cells 72 h after irradiation compared with those in non‑irradiated MC3T3‑E1 cells. Irradiation reduced colony formation and mineralization in a dose‑dependent manner in MC3T3‑E1 cells at 2 and 3 weeks after irradiation, respectively. Decreased levels of alkaline phosphatase activity and runt‑related transcription factor 2 expression were observed at 72 h post‑irradiation. In addition, irradiation‑induced apoptosis was accompanied by a decreased ratio of Bcl‑2/BAX protein and increased the activity of caspase‑3. By contrast, doxycycline (DOX)‑inhibited autophagy attenuated the decreased colony formation and mineralization, and aggravated the increased cell apoptosis in irradiated MC3T3‑E1 cells. Furthermore, the ratio of phosphorylated P38/P38 was observed to be higher following DOX treatment within 1 week of irradiation, which was reversed 2 weeks post‑irradiation. In conclusion, DOX‑inhibited autophagy aggravated X‑ray irradiation‑induced apoptosis at an early stage, but maintained cell proliferation and mineralization at a late stage in irradiated MC3T3‑E1 cells.