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Abstract: Background and Aim: The aim of this study is to investigate the impact of hepatitis B virus (HBV) S gene integration on serum hepatitis B surface antigen (HBsAg) levels in chronic hepatitis B with long‐term nucleos(t)ide analogue (NUC) therapy. Methods: Chronic hepatitis B patients who performed liver biopsy at baseline and treated with long‐term NUC therapy were recruited. The integration of HBV S gene in baseline liver biopsy specimen was detected by Alu polymerase chain reaction method. Serum HBsAg levels were measured at baseline and the second year and the fourth year after NUC therapy by Roche reagent, respectively. Serum HBsAg levels between HBV S gene integrated group and nonintegrated group were compared and analyzed. Results: Seventy patients were eligible for this study. Among them, 11 (15.7%) were found to have HBV S gene integration in their baseline liver biopsy specimens. Similar significant decrease of HBsAg levels was found in both integrated and nonintegrated groups (2.63 vs 2.65 log IU/mL, P = 0.478) after the first 2 years of NUC therapy. Thereafter, the decrease of HBsAg level from 2 to 4 years after therapy was largely unchanged in integrated group as compared with that of nonintegrated group (0.1 vs 2.53 log IU/mL, P = 0.002), with statistical difference. Conclusions: Serum HBsAg could be originated from the expression of the integrated HBV S gene in patients with S gene integration, which implicated the limitations when regarding HBsAg as a surrogate biomarker of covalently closed circular DNA activity and as an indicator of safe NUC discontinuation. [ABSTRACT FROM AUTHOR]