Mitochondrial respiratory oxidation is coupled with ATP synthesis. This coupling process can be broken by uncoupling protein (UCP), an integral membrane protein at mitochondrial inner membrane. Here, active rat UCP1 (rUCP1) was expressed in E. coli. Expression of rUCP1 can lower host’s growth rate. Immuno-electron microscopy proved expressed rUCP1 was mainly located on membrane. Purified rUCP1 was reconstituted into liposome and exhibited proton translocation activity. These results revealed eukaryotic UCP1 could be expressed in active form by prokaryotes and enable us to obtain enough amount of rUCP1 for structural study.