We investigated the activation mechanism of gene expression of lipocalin-type prostaglandin D synthase (L-PGDS) in human brain-derived TE671 cells. Reporter analyses of constructs carrying various lengths of the promoter region and intron 1 to 6, or 3'-untranslated region of the human L-PGDS gene demonstrated that one atypical E-box (aE-box) at +2569 in intron 4 was critical for transactivation of the gene. The aE-box inside the intron 4 functioned as an enhancer element in both directions and in a cell-type specific manner in TE671 cells. Yeast one-hybrid screening revealed that upstream stimulatory factor (USF) 1 bound to the aE-box. Expression of exogenous USF1 induced the endogenous L-PGDS expression in TE671 cells, whereas administration of USF1 siRNA suppressed L-PGDS expression. Binding of USF1 to the aE-box was confirmed by performing electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Furthermore, USF1-mediated transcriptional activation was dependent upon activator protein (AP)-2beta binding to the AP-2 element at position -98 in the proximal promoter region of human L-PGDS gene. These results indicate that L-PGDS gene expression in TE671 cells was activated by USF1 through the aE-box within intron 4 and cooperatively by AP-2beta in the promoter in a cell-type-specific manner.