Chromium (Cr) is an essential trace element for optimal insulin activity. It binds to the insulin receptor and potentiates many, and perhaps all, of its functions including the regulation of carbohydrate and lipid metabolism. The objective of this study was to investigate the effects of novel purified isomers of chromium histidinate (CrHis on protein levels of pancreatic glucose transporter protein 2 (GLUT-2), insulin receptor substrate (IRS-1) and glucagon-like peptide 1 receptor (GLP-1R). Forty-two Sprague–Dawley rats allocated to six groups (n=7). The rats were fed either 1: a standard diet (Control) or 2: a high-fat diet (HFD) or 3: a HFD with CrHis purified isomer 1 (CrHis1) or 4: a HFD with CrHis purified isomer 2 (CrHis2) or 5: a HFD with CrHis purified isomer 3 (CrHis3) or 6: a HFD with a mixture of three isomers of CrHis (CrHisM). Rats were fed the same amount of Cr (10 μg elemental Cr/kg BW/day), but from different sources of CrHis isomers. Rats fed a high-fat diet to induce insulin resistance. The administration of CrHis isomers showed an improvement in the HFD-enhanced pancreatic insulin levels. The insulin levels in the CrHis isomer groups were measured significantly lower than the HFD group (P<0.001).Pancreatic GLUT-2, IRS-1 and GLP-1R protein levels were markedly higher than those of the HFD group (P<0.001).Additionally, GLUT-2 and GLP-1R protein levels were higher in the CrHis1 group than the CrHis3 group (P<0.001), while no difference was found between the other isomers (P>0.05). As a result, it is thought that CrHis isomers or their mixture may exhibit a protective effect against HFD-induced insulin resistance in pancreatic tissue.