Patients infected with Human Immunodeficiency Virus (HIV), and other mammals infected with related lentiviruses, exhibit fatigue, altered sleep patterns and abnormal circadian rhythms. A circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) temporally regulates these functions in mammals. We found that a secretary HIV transcription factor, Transactivator of Transcription (Tat), resets the murine circadian clock, in vitro and in vivo, at clinically relevant concentrations (EC₅₀ = 0.31 nM). This effect of Tat occurs only during the subjective night, when N-Methyl-D-Aspartate (NMDA) Receptor (D-APV, 0.1 mM) and Nitric Oxide (NO) Synthase (L-NAME, 0.1 mM) inhibitors block Tat-induced phase shifts. Whole cell recordings of SCN neurons within the brain slice revealed that Tat did not activate NMDA receptors directly, but potentiated NMDA receptor currents through the enhancement of glutamate release. Consistent with this presynaptic mechanism, inhibitors of neurotransmission block Tat-induced phase shifts, such as tetrodotoxin (1 µM), tetanus toxin (1 µM), P/Q/N type-calcium channel blockers (1 µM -agatoxin IVA and 1 µM -conotoxin GIVA) and bafilomycin A1 (1 µM). Thus, the effect of Tat on the SCN may underlie lentiviral circadian rhythm dysfunction by operating as a disease-dependent modulator of light entrainment through the enhancement of excitatory neurotransmission.