The aim of this study was to evaluate the efficacy of different radioactive iodine (I-131) activities used for re-ablation, to compare various combinations of treatment activities, and to identify predictors of re-ablation failure in low- and intermediate-risk differentiated thyroid carcinoma (DTC) patients. The study included 128 consecutive low- and intermediate-risk patients with DTC with ablation failure after total thyroidectomy. Patient characteristics, T status, tumor size, lymph node involvement, postoperative remnant size on whole-body scintigraphy, serum thyroglobulin (Tg), thyroid-stimulating hormone (TSH), anti-Tg antibody (TgAb), and Tg/TSH ratio were analyzed as potential predictors of the re-ablation success. Re-ablation was successful in 113 out of 128 patients (88.3 %). Mean first I-131 activity was 2868 ± 914 MBq (77.5 ± 24.7 mCi) and mean second I-131 activity 3004 ± 699 MBq (81.2 ± 18.9 mCi). There was no association between the first, second, and cumulative activity with re-ablation treatment outcome. Treatment failure was associated with higher Tg levels prior to re-ablation (Tg2) (OR 1.16, 95 % CI 1.05-1.29, P = 0.003) and N1a status (OR 3.89, 95 % CI 1.13-13.41, P = 0.032). After excluding patients with positive-to-negative TgAb conversion, Tg2 level of 3.7 ng/mL predicted treatment failure with a sensitivity of 75.0 %, specificity of 80.5 %, and a negative predictive value of 97.1 %. Patients with positive-to-negative TgAb conversion had higher failure rates (OR 2.96, 95 % CI 0.94-9.29). Re-ablation success was high in all subgroups of patients and I-131 activity did not influence treatment outcome. Tg may serve as a good predictor of re-ablation failure. Patients with positive-to-negative TgAb conversion represent a specific group, in whom Tg level should not be used as a predictive marker of treatment outcome.