Objective To investigate the effect of different freezing thawing conditions on the stability of metronidazole sodium chloride injection in extreme cold environment. Methods The freezing process of metronidazole sodium chloride injection in extremely cold environment was simulated and appropriate conditions were given to melt. The effects of room temperature, 60 ℃ water bath thawing and freeze-thaw times on the content of metronidazole were investigated by HPLC, and the changes were analyzed by quality control chart. Results The linear range of metronidazole was 0.01～1.00 mg/ml and the relationship was acceptable, r=1.000, the average recovery was 101.03% (RSD=1.17%), which met the requirements of methodology. The samples were frozen at −20 ℃ and thawed in 60 ℃ water bath for 18 times with good stability. However, when thawed at room temperature, acicular crystals formed and the content decreased. Conclusion In extreme cold environment, the stability of metronidazole sodium chloride injection could be affected by freezing and thawing conditions. Therefore, in the use of Metronidazole and Sodium Chloride Injection, the number of freeze-thaw cycles should be minimized, and try to avoid thaw at room temperature.