It has been found that Ezrin is aberrantly expressed in some carcinomas and there is a relationship between the high level of Ezrin expression and metastatic potential of carcinomas. However, the molecular mechanisms underlying the regulation of the human ezrin gene transcription are not well understood. Transcriptional regulation regions of the human ezrin gene were examined by linking 5′-deletion mutants and site-directed mutagenesis of the 5′-flanking region to a luciferase reporter gene, and the basal promoter region and key DNA sequence elements (an Sp1 binding site, -75/-69; and an AP-1 binding site, -64/-58) involved in the expression of the human ezrin gene in lung cancer A549 cells were explored. Furthermore, electrophoretic mobility shift assay (EMSA) showed that DIG-ddUTP labeled DNA sequences containing ezrin key elements could bind nuclear extracts from lung cancer cells to form DNA-protein complex, and the bindings to Sp1 and AP-1 sites by rhSp1 and rhAP-1, respectively, were specific. Finally, the analysis of transient transfection of A549 cells by transcription factor expression vectors indicated that Sp1 and AP-1 (c-Jun/c-Fos heterodimer) could increase the human ezrin basal transcriptional activity significantly through the Sp1 and AP-1 binding sites, respectively. In addition, over-expression of Sp1, c-Jun or c-Fos could up-regulate Ezrin expression. The data suggested that the Sp1 and AP-1 binding sites were two key elements regulating ezrin gene basal transcriptional activity in lung cancer cells, and the corresponding transcription factors Sp1 and AP-1 were crucial for the transactivation.