Few studies have investigated the potential combined effects of multiple PCB congeners on diabetes. To address this gap, we used data from 1244 adults in the National Health and Nutrition Examination Survey (NHANES) 2003–2004. We used 1) classification trees to identify serum PCB congeners and their thresholds associated with diabetes; and 2) logistic regression to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) of diabetes with combined PCB congeners. Of the 40 PCB congeners examined, PCB 126 has the strongest association with diabetes. The adjusted OR of diabetes comparing PCB 126 > 0.025 to ≤ 0.025 ng/g was 2.14 (95% CI 1.30–3.53). In the subpopulation with PCB 126 > 0.025 ng/g, a lower PCB 101 concentration was associated with an increased risk of diabetes (comparing PCB 101 < 0.72 to ≥ 0.72 ng/g, OR = 3.3, 95% CI: 1.27–8.55). In the subpopulation with PCB 126 > 0.025&PCB 101 < 0.72 ng/g, a higher PCB 49 concentration was associated with an increased risk of diabetes (comparing PCB 49 > 0.65 to ≤ 0.65 ng/g, OR = 2.79, 95% CI: 1.06–7.35). This nationally representative study provided new insights into the combined associations of PCBs with diabetes.