Our purpose in this study was to assess the manganese (Mn) content in various organs in rats with or without glucose stimulation in vivo and in vitro by using magnetic resonance imaging (MRI) and polarized Zeeman atomic absorption spectrophotometry (PZAAS), respectively. MRI studies were performed in 12 rats using a 1.5-T MRI system. The rats were injected intravenously with saline (6 ml/kg) (saline-stimulated group, n = 6) or glucose (2.34 g/kg) (glucose-stimulated group, n = 6). Ten minutes after saline or glucose administration, MnCl₂ (0.02 mmol/kg) was injected intravenously, followed by 6 MRI studies at 8-min intervals. After the last MRI study, rats were killed, and the Mn concentrations in various organs were measured using PZAAS. There was a discrepancy between in vivo and in vitro measurements, which appeared to be due to the partial volume effect and/or the contribution of extracellular Mn. The Mn concentration in the pancreas, normalized to that in the liver in the glucose-stimulated group, increased significantly compared to that in the saline-stimulated group, suggesting that the influx of Mn into β cells in the pancreas increased in response to glucose stimulation. This study suggested that the measurement of the change in the Mn concentration due to glucose stimulation using PZAAS was effective for evaluating β-cell function in the pancreas.