Naturally occurring mutations in surface proteins of Hepatitis B virus (HBV) usually result in altered hepatitis B surface antigen (HBsAg) secretion efficiency. In the present study, we reported two conserved residues, M75 and M103 with respect to HBsAg, mutations of which not only attenuated HBsAg secretion (M75 only), but also suppressed HBV genome replication without compromising the overlapping p-gene product. We also found M75 and M103 can initiate truncated surface protein (TSPs) synthesis upon over-expression of full-length surface proteins, which may possibly contribute to HBV genome replication. However, attempts to rescue replication-defective HBV mutant by co-expression of TSPs initiated from M75 or M103 were unsuccessful, which indicated surface proteins rather than the putative TSPs were involved in regulation of HBV genome replication.