Women with gestational diabetes are at high risk for developing diabetes; post-partum weight loss may reduce the risk of diabetes. We evaluated the association of post-partum weight change with changes in glucose, insulin and homeostasis model assessment of insulin resistance in a subsample (n = 72) of participants from Diet Exercise and Breastfeeding Intervention (DEBI), a randomized pilot trial of lifestyle intervention for women with gestational diabetes. Glucose and insulin were measured fasting and 2 h after an oral glucose tolerance test at 6 weeks and 12 months post-partum. Women were categorized by weight change (lost > 2 kg vs. maintained/gained) between 6 weeks and 12 months post-partum. Compared with women who maintained or gained weight, women who lost > 2 kg experienced significantly lower increases in fasting glucose [age-adjusted means: 0.1 mmol/l (95% CI -0.03 to 0.3) vs. 0.4 mmol/l (95% CI 0.3-0.6); P < 0.01] and 2-h insulin [10.0 pmol/l (95% CI -56.9 to 76.9) vs. 181.2 pmol/l (95% CI 108.3-506.9); P < 0.01] and a significant reduction in 2-h glucose [-0.9 mmol/l (95% CI -1.4 to -0.3) vs. 0.3 mmol/l (95% CI -0.3 to 0.9); P < 0.01]. In multiple linear regression models adjusted for age, Hispanic ethnicity, medication use, meeting the Institute of Medicine's recommendations for gestational weight gain, breastfeeding and randomized group, a 1-kg increase in weight was significantly associated with increases in fasting and 2-h glucose (P < 0.05), but was not associated with insulin or homeostasis model assessment of insulin resistance. In women with gestational diabetes, modest post-partum weight loss may be associated with improvements in glucose metabolism.