Histidine decarboxylase (HDC) is the enzyme that catalyzes the conversion of histidine to histamine, a bioamine that plays an important role in allergic responses, inflammation, neurotransmission, and gastric acid secretion. Previously, we demonstrated that gastrin activates HDC promoter activity in a gastric cancer (AGS-E) cell line through three overlapping downstream promoter elements. In the current study, we used the yeast one-hybrid strategy to identify nuclear factors that bind to these three elements. Among eight positives from the one-hybrid screen, we identified Kruppel-like factor 4 (KLF4) (previously known as gut-enriched Kruppel-like factor (GKLF)) as one factor that binds to the gastrin responsive elements in the HDC promoter. Electrophoretic mobility shift assays confirmed that KLF4 is able to bind all three gastrin responsive elements. In addition, transient cotransfection experiments showed that overexpression of KLF4 dose dependently and specifically inhibited HDC promoter activity. Regulation of HDC transcription by KLF4 was confirmed by changes in the endogenous HDC messenger RNA by KLF4 small interfering RNA and KLF4 overexpression. We further showed that KLF4 inhibits HDC promoter activity by competing with Sp1 at the upstream GC box and also independently by binding the three downstream gastrin responsive elements. Taken together, these results indicate that KLF4 can act to repress HDC gene expression by Sp1-dependent and -independent mechanisms.