Intake of industrially produced trans-fatty acids (TFA) has been linked to increased risk of type 2 diabetes mellitus in observational studies. We investigated the causality of this association by examining if a high intake of TFA impairs measures of glucose homeostasis and induces intramuscular lipid deposition in abdominally obese women. In a double-blind, parallel dietary intervention study, 52 healthy but overweight postmenopausal women were randomized to receive either partially hydrogenated soybean oil (15 g/d TFA) or a control oil (mainly oleic and palmitic acid) for 16 weeks. Three markers of glucose homeostasis and 4 markers of lipolysis were derived from glucose, insulin, C-peptide, nonesterified fatty acid, and glycerol concentrations during a 3-hour frequent sampling oral glucose tolerance test. Intramuscular lipids were assessed by magnetic resonance spectroscopy. Forty-nine women completed the study. Insulin sensitivity (assessed by ISI(composite)), β-cell function (the disposition index), and the metabolic clearance rate of insulin were not significantly affected by the dietary intervention. Neither was the ability of insulin to suppress plasma nonesterified fatty acid and glycerol during oral glucose ingestion nor the intramuscular lipid deposition. In conclusion, high TFA intake did not affect glucose metabolism over 16 weeks in postmenopausal overweight women. A study population with a stronger predisposition to insulin resistance and/or a longer duration of exposure may be required for insulin sensitivity to be affected by intake of industrial TFA.