ABSTRACT Eg5 is one kind of kinesin motor that participates in various cellular processes, especially in mitosis. The tetrameric structure of Eg5 can crosslink the antiparallel microtubules and generate forces on microtubules to separate chromosomes through walking on the microtubules. Inhibition of the activity of Eg5 leads to cell cycle arrest and apoptosis. Thus, Eg5 is a potential therapeutic target in anticancer drug development. Two inhibitor‐binding sites have been discovered to date. The α2/L5/α3‐targeted inhibitors can inhibit the chemical cycle of Eg5. However, drug resistance to the α2/L5/α3‐targeted inhibitors is found in Eg5 with point mutations. The second inhibitor‐binding site is the pocket between α4 and α6 helices. The α4/α6‐targeted inhibitors can inhibit the activity of Eg5 with D130V and A133D mutations. However, the molecular mechanism of inhibition of α4/α6‐targeted inhibitors is still unclear. The effects of α4/α6‐targeted inhibitors on the Eg5‐microtubule interaction are investigated using molecular dynamics simulations. It is found that the binding of inhibitors can induce changes in the binding conformation of Eg5 on the microtubule. With inhibitors, the number of interactions formed between Eg5 and the microtubule are increased and the binding free energies are lowered. Therefore, α4/α6‐targeted inhibitors can enhance the Eg5‐microtubule interaction to inhibit the mechanical cycle of Eg5. It is also found that, Leu292, Leu293, and Tyr352 of Eg5 are key residues for the binding of inhibitors. These results provide molecular insight into the inhibition of α4/α6‐targeted inhibitors on the activity of Eg5.