Insulin gene expression is regulated by several islet-enriched transcription factors. However, MafA is the only beta cell-specific activator. Here, we show that MafA selectively induces endogenous insulin transcription in non-beta cells. MafA was also first detected in the insulin-producing cells formed during the second and predominant phase of beta cell differentiation, and absent in the few insulin-positive cells found in Nkx6.1(-/-) pancreata, which lack the majority of second-phase beta cells. These results demonstrate that MafA is a potent insulin activator that is likely to function downstream of Nkx6.1 during islet insulin-producing cell development.