Chronic exposure of pancreatic β-cells to elevated glucose reduces insulin gene promoter activity, and this is associated with diminished binding of two β-cell-enriched transcription factors, Pdx-1 and MafA. In this study using INS-1 β-cells, overexpression of MafA, but not Pdx-1, was able to restore expression of a human insulin reporter gene (−327 to +30 bp) suppressed by elevated glucose. At issue, however, was that MafA also markedly stimulated an insulin reporter gene (−230 to +30 bp) that was only marginally suppressed by glucose, suggesting that glucose-mediated suppression of the insulin promoter involved elements upstream of −230. Using serial truncations and minienhancer constructs of the human insulin promoter, the majority of glucose suppression was localized to regulatory elements between −327 and −261. Nuclear extracts from INS-1 cells exposed to elevated glucose had reduced binding activities to the A5/core (−319 to −307), and to a palindrome (−284 to −267) and an E box (−273 to −257, E3) contained within the Z element. The A5/core binding complex was determined to contain MafA, Pdx-1, and an A2-like binding factor. Two minienhancer constructs containing the A5/core were suppressed by glucose and strongly activated by MafA. Glucose-mediated suppression of the Z minienhancer was not attenuated by overexpression of MafA or Pdx-1. Site-directed mutation of the A5/core, palindrome, and E3 elements attenuated glucose-mediated suppression. These data indicate that glucose suppression of human insulin promoter activity in INS-1 cells involves reduced binding of MafA to the A5/core. Changes in nuclear factor binding to the Z element, which functions as a strong activator element in primary islets and a negative regulatory element in simian virus 40 or T antigen transformed β-cell lines, also participate in glucose suppression of insulin promoter activity.