Understanding adeno-associated virus (AAV) trafficking is critical to advance our knowledge of AAV biology and exploit novel aspects of vector development. Similar to the case for most DNA viruses, after receptor binding and entry, AAV traverses the cytoplasm and deposits the viral genome in the cell nucleus. In this study, we examined the role of the microtubule (MT) network in productive AAV infection. Using pharmacological reagents (e.g., nocodazole), live-cell imaging, and flow cytometry analysis, we demonstrated that AAV type 2 (AAV2) transduction was reduced by at least 2-fold in the absence of the MT network. Cell surface attachment and viral internalization were not dependent on an intact MT network. In treated cells at 2 h postinfection, quantitative three-dimensional (3D) microscopy determined a reproducible difference in number of intracellular particles associated with the nuclear membrane or the nucleus compared to that for controls (6 to 7% versus 26 to 30%, respectively). Confocal microscopy analysis demonstrated a direct association of virions with MTs, further supporting a critical role in AAV infection. To investigate the underling mechanisms, we employed single-particle tracking (SPT) to monitor the viral movement in real time. Surprisingly, unlike other DNA viruses (e.g., adenovirus [Ad] and herpes simplex virus [HSV]) that display bidirectional motion on MTs, AAV2 displays only unidirectional movement on MTs toward the nuclei, with peak instantaneous velocities at 1.5 to 3.5 μm/s. This rapid and unidirectional motion on MTs lasts for about 5 to 10 s and results in AAV particles migrating more than 10 μm in the cytoplasm reaching the nucleus very efficiently. Furthermore, electron microscopy analysis determined that, unlike Ad and HSV, AAV2 particles were transported on MTs within membranous compartments, and surprisingly, the acidification of AAV2-containing endosomes was delayed by the disruption of MTs. These findings together suggest an as-yet-undescribed model in which after internalization, AAV2 exploits MTs for rapid cytoplasmic trafficking in endosomal compartments unidirectionally toward the perinuclear region, where most acidification events for viral escape take place.