The impact of antithrombin replacement during extracorporeal membrane oxygenation (ECMO) in adults remains unclear. This work comprises a survey, showing that antithrombin is routinely supplemented in many Italian ECMO-Centers, and a retrospective analysis on 66 adults treated with veno-venous ECMO and unfractionated heparin at our Institution. Twenty-four to 72 h after the beginning of ECMO, antithrombin activity was ≤70% in 47/66 subjects and activated partial thromboplastin time (aPTT) ratio was <1.5 in 20/66 subjects. Activated partial thromboplastin time ratio <1.5 was associated not with lower antithrombin activity (61 ± 17 vs. 63 ± 22%; p = 0.983) but with higher circulating level of C-reactive protein (23 ± 8 vs. 11 ± 9 mg/dl; p < 0.001). In 34 subjects who received antithrombin concentrate, antithrombin activity increased (from 54 ± 9 to 84 ± 13%; p < 0.001); the proportion of subjects with aPTT ratio ≥1.5 increased (from 21/34 [62%] to 31/34 [91%]; p = 0.004); heparin dosage remained constant (from 19 ± 7 to 19 ± 6 IU/kg/h; p = 0.543); and C-reactive protein decreased (from 17 ± 10 to 13 ± 9 mg/dl; p = 0.013). Among those with aPTT ratio <1.5, aPTT ratio remained <1.5 in 3 out of 13 subjects. Antithrombin is frequently supplemented during veno-venous ECMO although low antithrombin activity does not constantly impede, and antithrombin replacement does not constantly ensure, reaching the target aPTT ratio. Inflammation possibly affects the individual response to heparin.