To investigate possible factors up-regulating the expression of UTROPHIN, potential regulatory elements in the promoter of the human UTROPHIN was predicted by P-match software and verified by EMSA and ChIP. The mechanism of EN1 regulation of the human UTROPHIN expression was evaluated by RNA interference and real-time PCR analyses. Two potential EN1 binding sites in UTROPHIN promoter region were predicted by P-Match software but only the second site was verified to interact directly with EN1 by EMSA and ChIP. The results from RNA interference and real-time PCR showed that the mRNA level of UTROPHIN increased in HeLa cells after EN1 was knockdowned by siRNA. It indicated that EN1 might be a negative regulatory factor for UTROPHIN. Our study suggested that UTROPHIN might be a new target for DMD therapy.