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Background Hepatitis B surface antigen (HBsAg) circulates in three isoforms, small (S)-, medium (M)-, and large (L)-HBsAg, which reflect specific steps of HBV life cycle and particle morphogenesis. In the setting of HBV/HDV co-infection, we assessed how HBsAg isoforms relate to markers of viral activity and liver injury in HDV-RNA-positive patients according to different clinical phenotypes.Methods This study included 366 viremic patients (n = 229 [62.6%] males; median age 56, IQR 46–62 years; n = 216 [59.0%] native Italians) from the HDV Describe study cohort. Serum S-, M-, and L-HBsAg levels were quantified by ELISA (Beacle Inc., Kyoto, Japan) and correlated with HDV-RNA (Robogene v2, Roboscreen GmbH, Leipzig, Germany), HBcrAg (Fujirebio, Tokyo, Japan), ALT, and liver stiffness measurement (LSM) (Fibroscan, Echosens, Paris, France). Patients’ clinical phenotypes included liver cirrhosis, chronic hepatitis D (CHD; HDV-RNA ≥ 3 Log IU/mL), and inactive/mild disease (HDV-RNA < 3 log IU/mL), as previously defined in Caviglia et al. Liver Int 2025.Results Median (IQR) HDV-RNA was 5.1 (3.4–6.0) log IU/mL and total HBsAg was 3331 (675–9055) ng/mL, while median (IQR) levels of S-, M-, L-HBsAg were 2686 (483–7234), 538 (92–1467) and 0.75 (0.10–4.50) ng/mL, respectively. Total, S-, M-, L-HBsAg showed positive correlations with HDV-RNA (rs=0.47, 0.47, 0.47, and 0.42, respectively; all p<0.001) and HBcrAg (rs=0.57, 0.56, 0.57, and 0.44, respectively; all p<0.001). Furthermore, total, S-, M-, and L-HBsAg levels were significantly higher in patients with ALT >40U/L compared to those with ALT ≤40 U/L (all p<0.001), whereas only L-HBsAg showed a correlation (albeit modest) with LSM (rs=0.16; p=0.007).Total, S, M-, L-HBsAg levels varied according to patients’ clinical phenotype, with the highest levels observed in high-viremic CHD, followed by cirrhosis and low-viremic CHD. Notably, in multivariate analysis, higher S-HBsAg levels were significantly associated with high-viremic CHD (OR=1.58, 95% CI 1.16–2.15; p=0.003), higher M-HBsAg with liver cirrhosis (OR=1.78, 95% CI 1.04–3.03; p=0.035), while lower L-HBsAg levels were associated with low-viremic CHD (OR=0.38, 95% CI 0.25–0.58; p<0.001).Conclusions Total HBsAg levels and HBsAg isoforms correlated with the extent of HDV replication and cytolytic activity. Notably, HBsAg isoforms showed distinct associations across clinical phenotypes, suggesting a differential regulation of HBV envelope proteins in HDV-driven liver disease.