Human herpesvirus 6 (HHV-6) infects and establishes latency in the central nervous system (CNS). Reactivation of latent HHV-6 has been associated with neurologic diseases including epilepsy and multiple sclerosis (MS). In vivo, HHV-6 has been localized to astrocytes and can infect human astrocytes in vitro, suggesting that this virus may have a tropism for glial cells and may affect glial cell function. An essential role of astrocytes in the CNS is active maintenance of the excitatory neurotransmitter glutamate. Dysregulation of glutamate has been implicated as a potential mechanism of disease in both epilepsy and MS. Both disorders have demonstrated elevated glutamate in CSF and may be associated with dysregulation of glutamate signaling, uptake, and metabolism. This study demonstrates dysregulation of glutamate uptake in human astrocytes infected with both variants of HHV-6, A and B, with differential effects of HHV-6 in acute and persistently infected cells. Whereas astrocytes acutely infected with HHV-6 demonstrated increased glutamate uptake, cells persistently infected with HHV-6A and HHV-6B demonstrated impaired glutamate uptake. Functional dysregulation of glutamate uptake was associated with early increases in mRNA and protein expression of the glial glutamate transporter EAAT-2 followed by a sustained decrease in mRNA expression in astrocytes infected with both HHV-6A and HHV-6B. Dysregulated glutamate uptake and transporter expression suggests a mechanism for dysregulation of glutamate levels in vivo and a potential mechanism for virus-associated neurologic disease.