Hepatitis B virus (HBV) is a small DNA virus with a compact genomic organization. All HBV proteins identified to date have been encoded by unspliced HBV RNAs. Spliced HBV RNAs have been described, but their functions are unknown. We show here that a singly spliced HBV RNA encodes a novel HBV protein in vivo. This HBV splice-generated protein (HBSP) corresponds to the fusion of a part of the viral polymerase and a new open reading frame that is created by the splicing event. In vivo, HBSP protein was found in HBV-infected liver samples, and anti-HBSP antibodies occurred in one-third of sera samples collected from chronic HBV carriers. In vitro, the ectopic expression of HBSP had no effect on viral DNA replication or transcription but induced cell apoptosis without a cell-cycle block. Overall, our results suggest that HBV has evolved a mechanism that directly modulates virus-cell interaction through RNA splicing.