AIM: To study the changes of mitochondrial membrane potential (△Ψm) in mouse thymocytes stimulated by dexamethasone (DEX). METHODS: Mouse thymocytes was stimulated by 1×10~-6 mol/L DEX. J-aggregate (~FL 2) and J-mononer (~FL 1) was measured by JC-1 staining flow cytometry at 0 h, 1 h, 3 h, 5 h, 7 h, 9 h, 11 h, 24 h and 27 h. △Ψm was calculated by (~FL 2/~FL 1). RESULTS: The mitochondrial mass of mouse thymocytes decreased rapidly in control group in vitro. ~FL 1 got platform at 5 h (913.38±70.11), then decreased till 27 h (622.80±22.81). In DEX group, there was no significant difference (P>0.05) compared with control group at 1 h and 3 h; while ~FL 1 in DEX group at 5 h (660.91±72.95) was significant lower (P<0.01) than that in control group, and decreased following culture time lapse till 27 h (309.70±53.35). The decrease in ~FL 2 was more obvious in both control and DEX group as the elongation of culture time. There was no significant difference for △Ψm between control and DEX group from 1 to 9 h. At 11 h, 24 h and 27 h, △Ψm in control groups were (256.41±21.59), (214.14±23.21) and (146.14±17.97), respectively, while △Ψm in DEX groups at the same time points were significantly lower than controls (P<0.01), they were (138.28±20.59), (111.61±29.67) and (72.92±17.86), respectively. CONCLUSIONS: During DEX-induced mouse thymocyte apoptosis process, mitochondrial mass decreases and mitochondria depolarization are early events, and the decrease in remaining mitochondrion △Ψm is a later event. According to these results, we assume that there is no necessary relationship between the mitochondrial mass decrease and △Ψm change of remaining mitochondrion.