Human immunodeficiency virus-1 (HIV) infection of the central nervous system promotes neuronal injury that culminates in HIV-associated neurocognitive disorders. Viral proteins, including transactivator of transcription (Tat), have emerged as leading candidates to explain HIV-mediated neurotoxicity, though the mechanisms remain unclear. Tat transgenic mice or neurons exposed to Tat, which show neuronal loss, exhibit smaller mitochondria as compared to controls. To provide an experimental clue as to which mechanisms are used by Tat to promote changes in mitochondrial morphology, rat cortical neurons were exposed to Tat (100 nM) for various time points. Within 30 min, Tat caused a significant reduction in mitochondrial membrane potential, a process that is regulated by fusion and fission. To further assess whether Tat changes these processes, fission and fusion proteins dynamin-related protein 1 (Drp1) and mitofusin-2 (Mfn2), respectively, were measured. We found that Drp1 levels increased beginning at 2 h after Tat exposure while Mfn2 remained unchanged. Moreover, increased levels of an active form of Drp1 were found to be present following Tat exposure. Furthermore, Drp1 and calcineurin inhibitors prevented Tat-mediated effects on mitochondria size. These findings indicate that mitochondrial fission is likely the leading factor in Tat-mediated alterations to mitochondrial morphology. This disruption in mitochondria homeostasis may contribute to the instability of the organelle and ultimately neuronal cell death following Tat exposure.