The nuclear receptor hepatocyte nuclear factor (HNF) 4α is involved in a transcriptional network and plays an important role in pancreatic β-cells. Mutations in the HNF4α gene are correlated with maturity-onset diabetes of the young 1. HNF4α isoforms result from both alternative splicing and alternate usage of promoters P1 and P2. It has recently been reported that HNF4α transcription is driven almost exclusively by the P2 promoter in pancreatic islets. We observed that transcripts from both P1 and P2 promoters were expressed in human pancreatic β-cells and in the pancreatic β-cell lines RIN m5F and HIT-T15. Expression of HNF4α proteins originating from the P1 promoter was confirmed by immunodetection. Due to the presence of the activation function module AF-1, HNF4α isoforms originating from the P1 promoter exhibit stronger transcriptional activities and recruit coactivators more efficiently than isoforms driven by the P2 promoter. Conversely, activities of isoforms produced by both promoters were similarly repressed by the corepressor small heterodimer partner. These behaviors were observed on the promoter of HNF1α that is required for β-cell function. Our results highlight that expression of P1 promoter-driven isoforms is important in the control of pancreatic β-cell function.