The frequent ACE insertion/deletion polymorphism (I/D) is, albeit inconsistently, associated with impaired glucose tolerance and insulin resistance. We recently observed an enhanced upregulation of ACE by elevated fat intake in GG-carriers of the I/D-surrogate rs4343 variant and therefore investigated its potential nutrigenetic role in glucose metabolism. In this nutritional intervention study 46 healthy and non-obese twin pairs consumed recommended low fat diets for 6 weeks before they received a 6-week high fat (HF) diet under isocaloric conditions. Intravenous glucose tolerance tests were performed before and after 1 and 6 weeks of HF diet. While glucose tolerance did not differ between genotypes at baseline it significantly declined in GG-carriers after 6 weeks HF diet (p = 0.001) with higher 2 h glucose and insulin concentrations compared to AA/AG-carriers (p = 0.003 and p = 0.042). Furthermore, the gene-diet interaction was confirmed in the cross- sectional Metabolic Syndrome Berlin Potsdam study (p = 0.012), with the GG- genotypes being significantly associated with prevalent type 2 diabetes for participants with high dietary fat intake ≥37% (GG vs. AA/AG, OR 2.36 [1.02–5.49], p = 0.045). In conclusion, the association between the rs4343 variant and glucose tolerance is modulated by dietary fat intake. The ACE rs4343 variant is a novel nutrient-sensitive type 2 diabetes risk marker potentially applicable for nutrigenetic dietary counseling.