Poxviruses are a group of large double-stranded DNA virus that replicate in the cytoplasm of the cell. The Orthopoxvirus genus includes variola virus, the etiological agent of smallpox, and vaccinia virus (VACV), the prototypical member of the genus. Cells infected with VACV display very little cell-cell fusion, however VACV mutants deleted for either the A56R or K2L gene display extensive cell-cell fusion. A56 and K2 interact with one another (A56/K2) and expression of both proteins is important for preventing cell fusion. VACV entry and fusion requires a multiprotein entry fusion complex (EFC) composed of at least eight proteins. In the absence of a functional EFC infected cell fusion does not occur even when the viruses lack either A56 or K2. A panel of recombinant VACVs was used to define protein interaction important for regulation of cell fusion. Affinity purification of A56, K2 and the EFC revealed an interaction between A56/K2 and the EFC. This interaction required expression of both A56 and K2 as A56 did not bind the EFC in the absence of K2 and vice versa. Interestingly, the ability to bind the EFC correlated with the inhibition of infected cell fusion by A56 and K2. Although the EFC contains eight proteins, only two entry proteins, A16 and G9, were important for binding A56/K2. Individually, A16 and G9 did not bind A56/K2; instead both A16 and G9 were needed for efficient interaction with A56/K2. A16 and G9 copurified with one another when expressed by transfection in uninfected cells, confirming that the two proteins bind to one another suggesting they directly interact within the EFC. To support a biological role for A56/K2 binding the EFC, cells expressing A56 and K2 were tested for infectivity as well as their ability to undergo cell-cell fusion. In both cases, cells expressing A56 and K2, but not individual expression of A56 or K2, showed reduced cell-cell fusion and virus entry. Collectively, these data support a model by which A56/K2 regulate infected cell fusion through an interaction with the viral EFC.