Pdx-1 is a key regulator of glucose-stimulated insulin gene transcription in beta-cells. The regulation of Pdx-1 in response to glucose has previously been associated with a remarkable shift in electrophoretic mobility on SDS-PAGE from 31 to 45kDa. This has been attributed to different post-translational modifications including phosphorylation, sumoylation or glycosylation. However, and in contrast with previous studies, we describe in this paper that Pdx-1 produced in Escherichia coli, by in vitro transcription/translation or exogenously expressed in eukaryotic cells, migrates with an apparent molecular mass of 45kDa despite a calculated mass of 31kDa. Moreover, we show that the migration of endogenous Pdx-1 obtained from a mouse beta-cell line as well as from human primary islets is not dependent on glucose concentration. Taken together, these data, validated by mass spectrometry techniques, establish that anomalous migration of Pdx-1 on SDS-PAGE does not result from post-translational modifications.