The effects of endogenous and exogenous nitric oxide on cancer cell mitochondrial membrane potential are studied by combining microplate fluorescence analyzer and special fluorescence probe rhodamine 123. Results show that the fluorescence intensity increases rapidly and keeps at a high level for a quite long period after nitric oxide donor is injected into the cells, while the fluorescence intensity of control group do not change significantly. Moreover, incubation with nitric oxide synthase inhibitor or nitric oxide donor for 24 hours prior rhodamine 123 loading can decrease or increase mitochondrial membrane potential, respectively. These results indicate that the increase of endogenous or exogenous nitric oxide can lift up the potential of mitochondrial membrane. It will be helpful to understand the role of nitric oxide in promoting cancer cell proliferation and metastasis at organelle level.