The hepatitis B surface antigen (HBsAg) is a vital serum marker for hepatitis B virus (HBV) infection. Amino acid (AA) substitutions in small hepatitis B surface protein (SHBs) are known to affect HBsAg level. However, how the genetic backbones of SHBs sequences would affect the roles of a specific AA substitution on HBsAg level remains unclear. In this study, we found that sI126 had a very high substitution detection rate of 17.54% (40/228) in untreated chronic hepatitis B cohort with subgenotype C2 HBV infection. Among different substitution types at sI126, the sI126T (N = 28) was found to be associated with significantly lower serum HBsAg level. Clone sequencing revealed that sI126T-harboring SHBs sequences had varied genetic backbones with zero to nine additional AA substitutions. Thus, we constructed 24 HBsAg expression plasmids harboring sI126T without (plasmid 1, P1) or with (P2-P24) additional AA substitution(s) and studied them in the HepG2 cells. The HBsAg levels were determined by both ELISA and Western blot. In vitro experiments showed that P1 significantly reduced HBsAg level and its secretion (p < .05), however, P2-P24 showed various extracellular and intracellular HBsAg levels. No significant differences were detected among the HBsAg mRNA levels of nine representative mutant plasmids. Our findings suggest that the modulation of HBsAg level by sI126T is affected by additional AA substitution(s) in the S region of HBV. The effects of AA combination substitutions in SHBs sequences on HBsAg levels are worthwhile for more attentions in terms of HBV biology and its clinical application.