Hepatitis B spliced protein (HBSP) encoded by a 2.2 kb singly spliced hepatitis B virus (HBV) pre-genomic RNA (spliced between positions 2447 and 489 nt) is involved in the pathogenesis of HBV infection, whereas the exact mechanism is far from being fully elucidated. In this study, a yeast two-hybrid system using HBSP as bait was employed to screen binding partners for HBSP from a human liver cDNA library. The interaction between HBSP and fibrinogen γ chain (FGG) was further confirmed in vitro using a GST pull-down assay and confirmed in vivo using a mammalian two-hybrid assay and co-immunoprecipitation. It was identified that this interaction is mediated by the N terminal 47 amino acid residues of HBSP. HBSP could inhibit fibrin polymerization, factor XIIIa-mediated fibrin cross-linking, adhesion of platelets to fibrinogen and ADP-stimulated platelet aggregation. However, the interaction-mediating fragment 1-47 of HBSP is not sufficient for the inhibitory activity on fibrinogen function. The findings suggested that HBSP may participate in the hemostatic abnormality in patients with HBV-related liver diseases.