In order to study the mechanism of hhlim gene transcriptional regulation, a series of deleted fragments of 5′ flanking region extending from +16 to －2 537 bp were subcloned into the pGL3-Basic vector respectively to identify the specific functional regions by detecting the luciferase activities. The results indicated that there was a silencer in the distal region of －2 537～－1 537 bp and an enhancer in proximal fragment of －253～－157 bp, respectively. Electrophoretic mobility shift assay showed that the patterns of shifted bands were different between the nuclear protein from differentiated C2C12 myotubes and undifferentiated C2C12 myoblasts when they bound to the region including the region of －253～－157 bp of hhlim gene. In addition, the results also showed that ET-1 and bFGF could not only significantly induce the hhlim gene expression in C2C12 cells but also activate the luciferase gene transcription promoted by －253～－157 bp regulatory region. It was suggested that hhlim gene was regulated by ET-1 and bFGF.