Human Krüppel-like factor 11 (hKLF11) has been characterised to both activate and inhibit human insulin promoter (hInsP) activity. Since KLF11 is capable to differentially regulate genes dependent on recruited cofactors, we investigated the effects of hKLF11 on cotransfected hInsP in both β-cells and non-β-cells. hKLF11 protein interacts with hp300 but not with hPDX1. Overexpressed hKLF11 stimulates PDX1-transactivation of hInsP in HEK293 non-β-cells, but confers inhibition in INS-1E β-cells. Both hKLF11 functions can be neutralised by the p300 inhibitor E1A, increased hp300 levels (INS-1E), dominant negative (DN)-PDX1 and by mutation of the PDX1 binding site A3 or the CACCC box. In summary, hKLF11 differentially regulates hInsP activity depending on the molecular context via modulation of p300:PDX1 interactions with the A3 element and CACCC box. We postulate that KLF11 has a role in fine-tuning insulin transcription in certain cellular situations rather than representing a major transcriptional activator or repressor of the insulin gene.