Diabetic retinopathy (DR), one of the major complications of diabetes, can cause blindness and reduce quality of life. Dyslipidemia is reported to be associated with DR, whereas arachidonic acid may have a protective effect against DR. We aimed to investigate the association of circulating n-3 and n-6 polyunsaturated fatty acids (PUFAs) with DR. In this cross-sectional study, 190 Japanese patients with type 2 diabetes were classified as no diabetic retinopathy (NDR), simple diabetic retinopathy (SDR), or proliferative diabetic retinopathy (PDR) including pre-proliferative diabetic retinopathy. Circulating fatty acids (FAs) were measured by gas chromatograph-mass spectrometry. Logistic regression analysis was performed to investigate the association between the levels of FAs and the presence of DR. The average age, body mass index and the duration of diabetes were 62.7 ± 12.1 years, 25.0 ± 4.5 kg/m2, and 9.8 ± 8.7 years, respectively. Twenty-seven patients were diagnosed with DR. Circulating levels of dihomo-gamma-linolenic acid (DGLA) in the NDR (n = 163), SDR (n = 13) and PDR (n = 14) groups were 28.3 ± 11.0 μg/mL, 24.4 ± 9.7 μg/mL, and 21.8 ± 6.2 μg/mL, respectively (p = 0.032). The logarithm of circulating DGLA levels was associated with the presence of DR after adjusting for covariates (OR of 1-unit increment: 0.79, 95% CI: 0.62–1.00, p = 0.049). Circulating DGLA was negatively associated with the presence of DR.