Background: Glycemic effects of the Women's Health Initiative (WHI) low-fat dietary intervention are unknown. Objective: Our objective was to analyze the effects of the WHI low-fat dietary intervention on serum glucose and insulin and insulin resistance up to 6 y after random assignment. Design: Postmenopausal WHI Dietary Modification trial intervention (DM-I) and comparison (DM-C) participants with blood measures at least at baseline and year 1 (n = 2263) were included. Anthropometric measures, dietary assessments, serum glucose and insulin concentrations, homeostasis model assessment of insulin resistance (HOMA-IR) measures, and quantitative insulin sensitivity check index (QUICKI) values were obtained at baseline, year 1, year 3, and year 6. Changes in measures were compared between groups at years 1, 3, and 6 overall and within stratified analyses. Results: Mean (±SD) differences in changes at year 1 between the DM-I and DM-C groups were as follows: glucose, -1.7 ± 17.9 mg/dL; insulin, -0.7 ± 5.1 μIU/mL; HOMA-IR, -0.2 ± 1.9; and QUICKI, 0.004 ± 0.019 (all P < 0.05). Similar findings resulted from repeated-measures analyses comparing the intervention and comparison groups over the 6 y. Whereas normoglycemic women at baseline had a decrease in glucose at year 1 that was 1.9 ± 17.2 mg/dL greater in the DM-I than in the DM-C group, diabetic women had an increase in glucose that was 7.9 ± 20.3 mg/dL greater in the DM-I than in the DM-C group (P for interaction <0.001). Conclusions: A low-fat diet was not significantly associated with adverse glycemic effects up to 6 y after random assignment in postmenopausal women. However, diabetic women experienced adverse glycemic effects of the low-fat diet. This trial is registered at clinicaltrials. gov as NCT00000611. © 2011 American Society for Nutrition.