In eukaryotic cells, intracellular and extracellular vesicle transport systems are ubiquitous and tightly linked. This process involves well-defined initiation and termination points, as well as mechanisms for vesicle recycling. During transport, cytoskeletal components serve as "roads" to prevent disordered vesicular movement and to ensure efficient transport, particularly through microtubules. Microtubules primarily facilitate the long-distance transport of vesicles. The dynamic nature of microtubule structure makes its stability sensitive to proteins, drugs, and post-translational modifications such as acetylation, which in turn regulate microtubule-dependent vesicular transport. Furthermore, motor proteins interact with microtubules and bind to cargoes their tail domains, driving vesicle transport along microtubules and determining the directionality of movement. To elucidate the detailed processes and mechanisms of microtubules-regulated long-distance vesicle transport, providing a comprehensive overview of current research in this area. This review provides an in-depth analysis of microtubule-mediated vesicle transport, emphasizing the molecular mechanisms involved. It examines vesicle transport between organelles, the impact of microtubule characteristics on this process, and the role of motor proteins in vesicle dynamics. Additionally, it summarizes diseases associated with abnormal microtubule-mediated vesicle transport, aiming to offer insights for the treatment of related conditions.