The viral Bcl-2 homolog (vBcl2) of Kaposi's sarcoma-associated herpesvirus (KSHV) displays efficient anti-apoptotic and anti-autophagic activity through its central BH3 domain, which functions to prolong the lifespan of virus-infected cells and ultimately enhances viral replication and latency. Independent on its anti-apoptotic and anti-autophagic activity, vBcl2 also plays an essential role in KSHV lytic replication through its amino-terminal 11-20 amino acids (aa). Here, we report a novel molecular mechanism of vBcl2-mediated regulation of KSHV lytic replication. vBcl2 specifically bound the tegument protein ORF55 through its amino-terminal 11-20aa, allowing their association with virions. Consequently, the vBcl2p peptide derived from the vBcl2 11-20aa effectively disrupted the interaction between vBcl2 and ORF55, inhibiting the incorporation of ORF55 tegument protein into virions. This study provides new insight of vBcl2's function in KSHV virion assembly that is separable from its inhibitory role of host apoptosis and autophagy.IMOPRTANCEKSHV, an important human pathogen accounting for a large percentage of virally caused cancers worldwide, has evolved a variety of stratagems for evading host immune responses to establish a life-long persistent infection. Upon viral infection, infected cells can go through a programmed cell death, including apoptosis and autophagy, that plays an effective role in anti-viral responses. To counter host response, the KSHV vBcl2 efficiently blocks apoptosis and autophagy to persist the lifespan of virus-infected cells. Besides its anti-programmed cell death activity, vBcl2 also interacts with ORF55 tegument protein for virion assembly in infected cells. Interestingly, the vBcl2p peptide disrupts the vBcl2-ORF55 interaction and effectively inhibits KSHV virion assembly. This study indicates that the KSHV vBcl2 harbors at least three genetically separable functions to modulate both host cell death signaling and virion production, and that the vBcl2p peptide can be developed as an anti-KSHV therapeutic application.