Abstract The ceramide family of lipids has been shown to play various important roles in both cell structure and signaling in a diverse array of cell types, including immune cells. However, very little is known regarding how ceramide affects the activation of dendritic cells (DCs) in response to viral infection. In this study, we demonstrate that a synthetic ceramide analogue (C8) stimulates DCs to increase the expansion of virus-specific CD8 T cells upon virus infection. Exogenously supplied C8 ceramide elevated the expression of DC maturation markers such as MHC class I and the co-stimulatory molecule B7-2 following infection with the immune suppressive Clone 13 strain of lymphocytic choriomeningitis virus (LCMV) in vitro. In addition, the ceramide analogue enhanced the production of IL-12 from DCs infected by the virus. Importantly, ceramide-conditioned, LCMV-infected DCs displayed a heightened activity to promote proliferation of virus-specific, CD8 T cells when compared to virus-infected DCs. Furthermore, locally instilled, but not systemically administered, ceramide analogue via intranasal inoculation significantly increased virus-reactive CD8 T cell responses in vivo. Collectively, these findings provide new insights into ceramide-mediated regulation of DC responses against virus infection and help us establish a foundation for novel immune-stimulatory therapeutics.