Mycoplasma synoviae (M. synoviae) is an avian pathogen responsible for respiratory disease and synovitis. The VlhA (variable lipoprotein hemagglutinin) family of surface adhesins plays a critical role in host cell attachment, yet the specific residues and structural determinants governing this interaction remain incompletely understood. In this study, we selected the C-terminal lipid-associated membrane hemagglutinin (LAM HA) domain within the VlhA family as the bait protein, based on its conserved C-terminal region. Yeast two-hybrid screening identified 18 LAM HA-interacting host proteins, and molecular docking pinpointed five residues (S83, R85, Y88, N124, K192) as the most frequent interaction hotspots. To assess their collective functional relevance, we constructed a combined deletion mutant lacking these five candidate residues. At the optimal pH of 6.0–6.5, wild-type LAM HA showed the highest titer (1:2), significantly exceeding that at pH 7.0–7.5 (no activity) and pH 5.0–5.5 (1:1) (p < 0.05), whereas the deletion mutant displayed a reduced titer (from 2 to 1; p = 0.05). Secondary structure analysis at the same pH revealed decreased α-helix content (from 7.90% to 7.57%), along with increased β-sheet (from 38.92% to 38.83%) and random coil (from 10.83% to 10.44%). Molecular dynamics simulations revealed that the deletion mutant exhibited elevated RMSF (from 2.83 ± 1.88 Å to 4.18 ± 2.65 Å) and Rg (from 40.48 ± 1.31 Å to 54.16 ± 4.47 Å) at pH 6.0–6.5, while RMSD showed a slight decrease (11.17 ± 1.33 Å vs. 12.14 ± 0.21 Å), collectively indicating compromised structural stability of the mutant. Collectively, these findings suggest that the LAM HA domain contributes to hemagglutination activity through pH-sensitive conformational stability and specific residue clusters—a property that may be relevant to M. synoviae colonization in the acidic microenvironments of the respiratory tract and synovial fluid during infection.