Purpose The changes of protein expression are involved in the pathophysiological process of acute high-altitude hypoxia-induced lung injury, which is mainly evaluated by western blot. There is no systematic study on the changes of internal control proteins as calibration loading amounts. Methods The low-pressure and low-oxygen chamber was used in the study to induce lunginjury at the altitude of 6000 meters for 8h、24h and 72h. Using HE staining to to confirm the establishment of the lung injury model. Western blot was used to detect the expression of different internal control proteins, including vinculin, α-tubulin, EIF5, β-actin, and GAPDH. At the same time, the total protein expression was detected by Coomassie Blue staining. Further, the bronchial epithelial cells (BEAS-2B) injury model and human umbilical vein endothelial cells (HUVECs) were induced by hypoxia 8h and 24h. And TUNEL staining was used to confirm the establishment of the cell injury model. Western blot was used to detect the expression of internal control proteins as same as that in – vitro model. The total protein expression was detected by Coomassie Blue staining. Results Acute hypoxic models of 8h, 24h, and 72h were successfully established in lung tissue, demonstrating consistent total protein expression and stable levels of internal reference proteins vinculin, α-tubulin, EIF5, and β-actin. The expression of GAPDH was elevated in the 8h, 24h, and 72h groups compared to the control group without statistical significance at 8 h and 24 h but significantly increased at 72h. Similarly, hypoxic models of 8h, 24h, and 48h were successfully established in BEAS-2B and HUVECs with consistent total protein expression. In BEAS-2B cells, the internal reference proteins β-actin and GAPDH exhibited consistent expression with the normoxic control group while vinculin, α-tubulin, and EIF5 showed significantly reduced expression at hypoxic conditions for a duration of up to 24h. In HUVECs, vinculin, α-tubulin and β-actin also maintained consistent expression with the normoxic control group while EIF5 and GAPDH showed significant reduction at the hypoxic time point of 8h and increased expression at 48h. Conclusion Acute hypoxic exposure induces lung tissue injury, and the proteins expression of internal reference proteins vinculin, α-tubulin, EIF5，and β-actin is stable, making the propriate choices for internal references in Western blotting. Additionally, differential protein expression levels for internal reference proteins vinculin, α-tubulin, EIF5, β-actin and GAPDH were observed in BEAS-2B and HUVEC cells when applying Western blotting to the most important lung tissue models invitro, BEAS -2B and HUVECs, to induce hypoxia-induced injury.