Objective: Chromium has gained popularity as a nutritional supplement for diabetic patients. This study evaluated the effect of chronic administration of a chromium complex of D-phenylalanine (Cr(D-phe)3) on glucose and insulin tolerance in obese mice. The study tested the hypothesis that Cr(D-phe)3 suppresses endoplasmic reticulum (ER) stress and insulin resistance in these animals. Methods and Procedures: C57BL lean and ob/ob obese mice were randomly divided to orally receive vehicle or Cr(D-phe)3 (3.8 μg of elemental chromium/kg/day) for 6 months. Insulin sensitivity was evaluated by glucose and insulin tolerance tests. Protein levels of phosphorylated pancreatic ER kinase (PERK), α subunit of translation initiation factor 2 (eIF2α) and inositol-requiring enzyme-1 (IRE-1), p-c-Jun, and insulin receptor substrate-1 (IRS-1) phosphoserine-307 were assessed by western blotting. In vitro ER stress was induced by treating cultured muscle cells with thapsigargin in the presence or absence of Cr(D-phe)3. Results: ob/ob mice showed poor glucose and insulin tolerance compared to the lean controls, which was attenuated by Cr(D-phe) 3. Markers of insulin resistance (phospho-c-Jun and IRS-1 phosphoserine) and ER stress (p-PERK, p-IRE-1, p-eIF2α), which were elevated in ob/ob mice, were attenuated following Cr(D-phe)3 treatment. Chromium treatment was also associated with a reduction in liver triglyceride levels and lipid accumulation. In cultured myotubes, Cr(D-phe) 3 attenuated ER stress induced by thapsigargin. Discussion: Oral Cr(D-phe)3 treatment reduces glucose intolerance, insulin resistance, and hepatic ER stress in obese, insulin-resistant mice. © 2008 The Obesity Society.