This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β-cells. INS-1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1–1 µM) for 12 h. The phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose-dependent manner. SPIO labeling (200 µg Fe ml−1) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose-stimulated phosphorylation of ERK1/2 and AKT and insulin-stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS-1 cells, through modulation of the glucose- or insulin-induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis. Copyright © 2012 John Wiley & Sons, Ltd.