BACKGROUND: Diet interventions have shown effectiveness in improving diabetes risk factors; however, little is known about whether the effects of diet intervention are different according to genetic susceptibility. OBJECTIVE: We examined interactions between weight-loss diets and the genetic risk score (GRS) for diabetes on 2-y changes in markers of insulin resistance and β cell function in a randomized controlled trial. DESIGN: Data from the Preventing Overweight Using Novel Dietary Strategies (POUNDS LOST) trial were analyzed. A GRS was calculated on the basis of 31 diabetes-associated variants in 744 overweight or obese nondiabetic adults (80% white Americans). We assessed the changes in insulin resistance and β cell function over the 2-y intervention. RESULTS: Dietary protein significantly interacted with the diabetes GRS on fasting insulin, glycated hemoglobin (HbA1c), the homeostasis model assessment of β cell function (HOMA-B), and the homeostasis model assessment of insulin resistance (HOMA-IR) at 2 y in white Americans (P-interaction = 0.02, 0.04, 0.01, and 0.05, respectively). The lower GRS was associated with a greater decrease in fasting insulin (P = 0.04), HbA1c (P = 0.0001), and HOMA-IR (P = 0.02), and a lesser increase in HOMA-B (P = 0.004) in participants consuming a low-protein diet. Participants with a higher GRS might have a greater reduction in fasting insulin when consuming a high-protein diet (P = 0.03). CONCLUSIONS: Our data suggest that individuals with a lower genetic risk of diabetes may benefit more from consuming a low-protein weight-loss diet in improving insulin resistance and β cell function, whereas a high-protein diet may be more beneficial for white patients with a higher genetic risk. This trial was registered at clinicaltrials.gov as NCT00072995.