The rapid onset of potentially lethal neuroinflammation is a defining feature of viral encephalitis. Glial cells are ideally positioned to respond to invading pathogens of the central nervous system (CNS) and produce key inflammatory mediators following viral infection. However, the mechanisms by which resident CNS cells perceive such challenges have not been defined. Recently, several cytosolic pattern recognition receptors, including retinoic acid-inducible gene I (RIG-I) and DNA-dependent activator of IFN regulatory factors (DAI), have been described that appear to function as intracellular sensors of RNA and DNA viruses, respectively. Interestingly, recent studies suggest that RIG-I may also be able to recognize DNA pathogens in a polymerase III-dependent manner. However, little is known regarding the expression of these novel intracellular viral sensors or their role in the CNS. In the present study, we demonstrate that microglia and astrocytes constitutively express detectable levels of both RIG-I and DAI and their downstream adaptor molecules. In addition, we show that expression of RIG-I and DAI by glial cells is elevated following infection with the model neurotropic RNA virus, vesicular stomatitis virus (VSV), and/or DNA viruses including the neurotropic virus herpes simplex virus-1 (HSV-1), or specific synthetic ligands for these viral sensors. Importantly, these specific ligands elicit inflammatory mediator production by both microglia and astrocytes, and targeted knockdown of RIG-I or DAI attenuates such responses following RNA or DNA virus exposure, respectively, and limits the production of soluble neurotoxic mediators by virally challenged cells. Interestingly, glial inflammatory responses to the DNA virus HSV-1 were also dependent on the expression of RIG-I and the activity of polymerase III, while glial responses to the RNA virus VSV required the expression of RIG-I but were DAI and polymerase III independent. These studies demonstrate that RIG-I and DAI play a critical role in the recognition of viral pathogens by resident CNS cells and suggest that these novel intracellular pattern recognition receptors may underlie the damaging inflammation and neuronal cell death associated with acute neurotropic viral infections.