Ehrlichia chaffeensis is a Gram-negative, obligate intracellular bacterium which causes the tick-borne disease human monocytic ehrlichiosis. In vertebrates, E. chaffeensis replicates in monocytes and macrophages. However, no clear cell or tissue tropism has been defined in arthropods. Our group identified two host genes that control E. chaffeensis replication and infection in vivo in Drosophila, Uridine cytidine kinase and separation anxiety. Using the UAS-GAL4 RNAi system, we generated F1 flies (UAS-gene of interest RNAi x tissue-GAL4 flies) that have Uck2 or san silenced in ubiquitous or tissue-specific fashion. When Uck2 or san were suppressed in the hemocytes or in the fat body, E. chaffeensis replicated poorly and caused significantly less severe infections. Silencing of these genes in the eyes, wings, or the salivary glands did not impact fly susceptibility or bacterial replication. Our data suggest that in Drosophila, E. chaffeensis replicates within the hemocytes, the insect homolog of mammalian macrophages, and in the fat body, the liver homolog of mammals.