The functional significance of a non-conserved cysteine residue (C86) proximal to the interfacial region of Rhipicephalus microplus triosephosphate isomerase (RmTIM) was investigated through systematic substitution with aspartic acid (C86D), lysine (C86K), and alanine (C86A) via site-directed mutagenesis. Kinetic analyses revealed substantial perturbations in enzymatic parameters for the C86D and C86K variants, with marked alterations in maximal velocity (Vmax), substrate affinity (Km), catalytic turnover (kcat), and catalytic efficiency (kcat/Km). Thermodynamic destabilization was universally observed across all mutants via Protein Thermal Shift assays, with reductions in melting temperature (Tm) ranging from 15.7 to 27.1 °C relative to the wild-type enzyme (Wt-TIM). Chemical denaturation studies employing guanidine hydrochloride demonstrated heightened susceptibility to unfolding in all mutants, with destabilization profiles following the order: C86K > C86D > C86A. Computational structural analyses elucidated molecular mechanisms underlying these perturbations. Disruption of a putative salt bridge between residues D49 and K18 was predicted in the C86K mutant, potentially destabilizing the dimeric interface. Comparative free energy calculations (ΔG) further corroborated these findings: Wt-TIM exhibited a ΔG of −21.2 kcal/mol and an interfacial contact area of 1604.8 Å2, indicative of robust dimeric stabilization. In contrast, the C86K mutant displayed diminished stability (ΔG = −16.0 kcal/mol) despite an expanded interface (1615.6 Å2), suggesting compromised packing efficiency. These observations imply that C86, while not directly conserved, plays a critical structural role in maintaining interfacial integrity and catalytic competence. The pronounced destabilization and kinetic impairment observed in the C86K variant highlight the residue's significance in RmTIM functionality. This residue-specific destabilization strategy may aid in the rational design of acaricidal compounds targeting interfacial regions of RmTIM, taking advantage of structural vulnerabilities produced by non-conserved residues. © 2025