Background: There is inconsistent epidemiologic evidence for an association between tissue levels of n-3 polyunsaturated fatty acids (PUFAs) and the risk of type 2 diabetes. The purpose of the present study was to investigate whether erythrocyte levels of n-3 PUFAs are negatively associated with the risk of type 2 diabetes and correlated with levels of glucose and HbA1c in Koreans. Methods: A total of 130 patients with type 2 diabetes and 260 age- and sex-matched controls participated in this study. Results: The risk of type 2 diabetes was negatively associated with erythrocyte levels of 22:5n3 and 22:6n3, which were negatively correlated with HbA1c levels after adjusting for age, sex and body mass index. Additionally, the risk of type 2 diabetes was negatively associated with erythrocyte levels of 18:0 and 20:4n6, but positively associated with erythrocyte levels of 16:0, 18:1n9, 18:3n3, 18:2n6, 18:3n6, n-6/n-3 PUFA ratio, 18:1t and 18:2n6t, and Δ-6 desaturase (18:3n6/18:2n6) and Δ-9 desaturase (18:1n9/18:0) activities. Conclusions: The risk of type 2 diabetes was negatively associated with erythrocyte levels of n-3 PUFA, which were negatively correlated with HbA1c levels in Koreans, suggesting that n-3 PUFAs might reduce the risk of type 2 diabetes in Asians.