The vaccinia viral protein A27 on mature viruses specifically interacts with heparan sulfate for cell surface attachment. A27 associates with viral membrane protein A17 to anchor to the viral membrane, and yet the specific interaction between A27 and A17 remains largely unclear. To uncover the active binding sites and underlying binding mechanism, we expressed and purified N- and C-terminal fragments, A17-N (18-50) and A17-C (162-203). By SPR, the binding affinity of A27/A17-N (KA = 3.40 × 108 M−1) was determined to be approximately 3 orders of magnitude stronger than that of A27/A17-C (KA = 3.40 × 105 M−1), indicating that A27 prefers to interact with A17-N rather than A17-C. By NMR, despite the disordered nature of A17-N, we demonstrated that the A27-A17 interaction is mediated by a specific and cooperative binding mechanism via two active binding sites, namely 32SFMPK36 (denoted as F1 binding) and 20LDKDLFTEEQ29 (F2), where F1 has a stronger binding affinity and was more resistant to acidic conditions than F2. Furthermore, A27 mutant proteins that retained partial activity to interact with the F1 and F2 sites of A17 protein were packaged into MV particles at a reduced level, demonstrating the F1/F2 site interaction plays a critical role in vivo. Combined with site-directed mutagenesis data, we thus established a computer model to explain the specific A27-A17 binding mechanism.