<p>Microtubules are crucial components of the mitotic spindle, essential in chromosome segregation during cell division. EG5, a kinesin motor protein, has emerged as a critical player in this process by promoting the separation of sister chromatids. Dysregulation of EG5 function is associated with tumorigenesis, making it a promising target for cancer therapeutics. Hence, understanding EG5’s molecular mechanisms is a key to developing better therapies with fewer side effects. Here, we investigate the mechanisms by which EG5 interacts with microtubules and how this interaction enhances its motor activity. Utilizing computational methods, we probe the role of microtubule binding in the allosteric regulation of EG5 dynamics. Our results demonstrate that microtubule binding significantly enhances EG5’s dynamic flexibility and motor activity, while inhibitors targeting distinct allosteric sites disrupt this interaction. These insights provide a molecular framework for the rational design of EG5-targeted inhibitors, with potential implications for anticancer drug development.</p>