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Abstract Vaccinia virus enters host cells through an 11-protein entry fusion complex (EFC) that operates by a mechanism distinct from those of canonical viral fusion systems. Understanding how this multiprotein complex mediates membrane fusion is crucial for elucidating poxvirus entry and identifying potential antiviral targets. Here, we determined the NMR structure of a truncated J5 protein, (J5 2-68 ). To further dissect the functional determinants of J5, we generated recombinant vaccinia viruses expressing various J5 mutants, including substitutions in conserved residues, alterations of exposed charged residues, and chimeric constructs between vaccinia J5 and its orthologous AMV232 gene from an entomopoxvirus. Functional analyses revealed that residues 90-110 and the conserved P 38 YYCWY 43 motif are indispensable for maintaining EFC integrity and promoting membrane fusion. Together, we define the structural and functional elements of J5 that are essential for poxvirus entry and advance our understanding of the unique membrane fusion mechanism employed by poxviruses. Importance Vaccinia virus enters host cells through an eleven-protein entry fusion complex (EFC) that is mechanistically distinct from canonical viral fusion systems. Understanding how this multiprotein machinery mediates membrane fusion is essential for elucidating poxvirus entry mechanisms and for developing antiviral strategies. Here, we determined the NMR structure of the J5 ectodomain and generated a series of recombinant vaccinia viruses carrying J5 mutations. Functional analyses identified two regions, residues 90-110 and the conserved P 38 YYCWY 43 motif, as essential for EFC stability and membrane fusion. These findings provide the first structure-function framework for J5, a core component of the EFC, and reveal key determinants required for complex integrity and viral entry.