We previously demonstrated that the transcription factor Pax4 is important for beta-cell replication and survival in rat islets. Herein, we investigate Pax4 expression in islets of non-diabetic and diabetic donors, its regulation by mitogens, glucose and the incretin GLP-1 and evaluate its effect on human islet proliferation. Pax4 expression was increased in islets derived from Type 2 diabetic donors correlating with hyperglycaemia. In vitro studies on non diabetic islets demonstrated that glucose, betacellulin, activin A, GLP-1 and insulin increased Pax4 mRNA levels. Glucose-induced Pax4 expression was abolished by the inhibitors LY294002, PD98050 or H89. Surprisingly, increases in Pax4 expression did not prompt a surge in human islet cell replication. Furthermore, expression of the proliferation marker gene Id2 remained unaltered. Adenoviral-mediated expression of human Pax4 resulted in a small increase in Bcl-xL expression while Id2 transcript levels and cell replication were unchanged in human islets. In contrast, overexpression of mouse Pax4 induced human islet cell proliferation. Treatment of islets with 5-Aza-2'-deoxycytidine induced Pax4 without stimulating Bcl-xL and Id2 expression. Human Pax4 DNA binding activity was found to be lower than that of the mouse homologue. Thus, human pax4 gene expression is epigenetically regulated and induced by physiological stimuli through the concerted action of multiple signalling pathways. However, it is unable to initiate the transcriptional replication program likely due to post-translational modifications of the protein. The latter highlights fundamental differences between human and rodent islet physiology and emphasizes the importance of validating results obtained with animal models in human tissues.