Leptin, a 16-kDa hormone produced by mature adipocytes, has been shown to regulate the hibernation of mammals. In this study, the leptin gene sequences of both hibernating (Rhinolophus ferrumequinum) and non-hibernating (Rousettus leschenaultii) bats were determined, and the leptin proteins from these two different species of bats were expressed in Escherichia coli for the first time. Results showed that the amino acid sequence of the leptin protein from hibernating bats had a lower degree of identity than that from non-hibernating bats to those of several non-hibernating mammals. The leptin protein of hibernating bats had a stronger growth inhibitory effect on 3T3-L1 cells than that of non-hibernating bats. Structural modeling revealed that the structures of the receptor binding site III, which is critical for signal transduction, of the two bat leptins were very different. Similar to the human leptin, the leptin protein of non-hibernating bats was predicted to have a random loop, whereas that of hibernating bats had a helical structure in this region. This observation provided a clue as to the differential effects of the two different leptins on 3T3-L1 cells.