Kinesin-5 drives separation of microtubules and organizes the mitotic spindle via its motor domains. More than one component within Kinesin-5 has been implicated in its motile ability; these are Loop-5, the necklinker and the cover neck. In all studies, measured changes in structure, or their implied dynamic motion, were deemed key to the role of these components. Unaddressed is whether these protein components chemically direct mechanical output. Herein, we show that specific side-chain chemistry of Loop-5 must be conserved for productive Kinesin-5 motility. Six substitutions of Loop-5 residues were created in the Eg5 dimer by mutagenesis, and their microtubule-gliding velocities were measured. Mutant Eg5 gliding velocities ranged from nearly equivalent to wildtype to a complete loss of motility. There were key differences between the mutations in the N- versus C-terminus of Loop-5. Substitutions near the N-terminus retained the ability to glide microtubules; alteration of gliding velocity paralleled changes in microtubule-stimulated catalytic rates. In contrast, nonconservative substitutions near the C-terminus of Loop-5 bound microtubules in rigor despite having robust ATPase activity. Collectively, these results suggest the integrity of the active site remains intact and communication between the active site and microtubule site is not compromised. Mechanical output is challenged, however. Therefore, we conclude that side-chain interactions of these C-terminal residues with the surrounding protein matrix are required for the terminal step in Eg5 mechanotransduction. Given our kinetic data, we speculate that aberrant interactions may result in changes in force and/or coordination of the two motor domains in the dimer, rendering Eg5 incapable of motility. This work is funded by the support of the National Institutes of Health (R01 GM097350 S.K.;P20GM103424 and 5G12RR026260 T.H.) and the LSU School of Graduate Studies (R.B.).