We sought to determine whether a G→A variant at position −30 of the β-cell promoter of the glucokinase (GCK) gene observed to be present more frequently in Japanese-American men with impaired glucose tolerance (IGT) than in Japanese-American men with normal glucose tolerance (NGT) is associated with impaired β-cell function. We studied 125 unrelated Japanese-American men (aged 46–74 years; mean 61 ± 0.5) who were nondiabetic by a 75-g oral glucose tolerance test (OGTT) (65 had NGT and 60 had IGT). The presence of the −30 β-cell GCK gene promoter variant was determined by single-strand conformation polymorphism analysis. β-cell function was assessed using the ratio of the incremental response in immunoreactive insulin (IRI) to that of glucose during the first 30 min of the OGTT (Δ IRI[30 min−0 min]/Δglucose[30 min−0 min]) performed at baseline and at 5 years of follow-up. β-cell function adjusted for basal IRI ([Δ IRI[30 min−0 min]/Δ glucose[30 min⁻⁰ min]]/basal IRI; the relative insulin response) was also evaluated. At baseline, the −30 β-cell GCK gene promoter variant was present in 15.4% of subjects with NGT vs. 38.3% of subjects with IGT (P < 0.01). Fasting IRI did not differ between groups. At baseline, ΔIRI[30 min⁻⁰ min]/Δglucose[30 min⁻⁰ min] was significantly lower in subjects with the promoter variant (57 × 10⁻⁹ to 95 × 10⁻⁹ to 95 × 10⁻⁹] vs. 77 × 10⁻⁹ [55 × 10⁻⁹ to 128 × 10⁻⁹]; median [interquartile range]; P < 0.01) as was the relative insulin response (0.97 [0.70–1.24] vs. 1.37 [0.95–2.03] l/mmol; P < 0.0005). Similarly, at 5 years of follow-up, ΔIRI[30 min−0 min]/Δglucose[30 min−0 min] and the relative insulin response were significantly reduced in the group with the variant. In the subgroups of subjects with IGT at baseline, IGT at 5 years, and NGT at 5 years, the relative insulin response was significantly lower in those with the variant. We conclude that the −30 β-cell GCK gene promoter variant is associated with reduced β-cell function in middle-aged Japanese-American men and may contribute to the high risk of abnormal glucose tolerance in this population.