Objective A relationship between thyroid and non-organ-specific autoimmunity could be relevant for Graves’ orbitopathy (GO), which affects connective tissue. We investigated the association between GO and anti-nuclear antibodies (ANAs). Methods Retrospective investigation was conducted in 265 patients with Graves’ disease (GD), 158 with and 107 without GO. Primary outcome was: prevalence of ANAs in GO vs no-GO. Secondary outcomes were: (1) relationship between ANAs and GO features; (2) prevalence of ANAs in GD compared with non-autoimmune hyperthyroidism [(78 patients with toxic nodular goiter (TNG)]; (3) distribution of ANA patterns. Results ANAs were detected in 212 (80%) GD patients, but prevalence did not differ between GO (79.7%) and no-GO (80.3%). Higher ANA titers (1:160) were more common in GO (51.5 vs 38.3%), but only nearly significantly (OR 0.5; 95% CI: 0.3–1; P = 0.059). Proptosis was lower in ANA-positive patients (mean difference: − 1.4 mm; 95% CI from − 2.5 to − 0.3; P = 0.011), in whom nearly significantly lower CAS (Mann–Whitney U: 1.5; P = 0.077) and eyelid aperture (mean difference: − 0.9 mm; 95% CI from − 2 to 0; P = 0.062) were observed. Prevalence of ANAs in GD was lower than in TNG (80 vs 91%; OR 0.3; 95% CI: 0.1–0.9; P = 0.028), but nuclear speckled pattern was more frequent (OR 22.9; 95% CI 1.3–381.3; P = 0.028). Conclusions Although ANAs are not more frequent in GO, they seem to exert a protective role on its severity and on development of GD. A switch of T cell population in ANA-positive patients, resulting in a different phenotype, may be responsible. Further studies are needed to investigate the mechanisms.