Objective The myocardial injury was induced by hypobaric hypoxia through regulating the expression of various proteins. The expression of proteins was mainly detected by western blot, but the selection of internal reference proteins and their variations have not been systematically studied. Methods Myocardial injury was induced in a low-pressure, low-oxygen chamber simulating an altitude of 6000 m, for 24 and 72 h. Establishment of the myocardial injury model was confirmed by hematoxylin eosin(HE) staining. Expression levels of internal control proteins, including vinculin, α-tubulin, eukaryotic translation initiation factor-5 (EIF5), β-actin, glyceraldehyde-3phosphate dehydrogenase (GAPDH), cyclophilin B, and cofilin, were detected by Western Blot and total protein expression was detected by Ponceau S and Coomassie Blue staining. An adult mouse cardiomyocytes (AMCMs) injury model was induced by hypoxia for 12 and 24 h and confirmed by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL staining). Internal control proteins were detected by Western Blot, as in the in vivo model, and total protein expression was detected by Ponceau S and Coomassie Blue staining. Results A myocardial injury model was established by hypobaric hypoxia for 24 and 72 h, the total protein expression levels remained consistent. The expression of internal control proteins including vinculin, EIF5, β-actin, cyclophilin B, and cofilin was consistent between the control and model groups. Expression levels of α-tubulin were similar in the plain control and 24 h hypobaric hypoxia group, but were significantly lower in the 72 h hypobaric hypoxia group compared with the plain control group. GAPDH expression was significantly higher in the 24 and 72 h hypobaric hypoxia groups than in the plain control group. An AMCM injury model was established by hypoxia for 12 and 24 h. Total protein levels and expression levels of the internal control proteins EIF5 and β-actin were consistent, but vinculin, α-tubulin, GAPDH, cyclophilin B, and cofilin expression levels were higher in both hypoxia groups compared with the normoxic control group. Conclusions EIF5 and β-actin may be the suitable loading control proteins for studies of hypobaric hypoxia induced myocardial injury using Western Blot. Total protein is also a good choice for hypobaric hypoxia studies.