Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2alpha-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking region of the PTEN gene showed that the region between -1175 and -1077 from the translational initiation site, which contains the AP-2alpha binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2alpha is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2alpha diminished the enhancement of AP-2alpha and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2alpha also results in the induction of PTEN transcription in AP-2alpha-negative colon cancer cells. Additionally, GM3 induced AP-2alpha-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2alpha transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene.