Defective viral genomes (DVGs) are truncated genomes that have been observed during multiple RNA virus infections, including in vitro Ebola virus (EBOV) infection. Copy back DVGs, which have been associated with viral persistence and immunostimulatory activity, are made of two complementary copies of the 5’ trailer region of the genome. As DVGs have previously been detected in Vero E6 cells persistently infected with EBOV, we hypothesized that DVGs may also accumulate during viral replication in an EBOV-infected host. We have analyzed sequencing data from EBOV-infected non-human primate (NHP) serum and tissues and demonstrated that DVGs accumulate during the acute phase of EBOV infection. To investigate the effect of DVGs on EBOV infection, we generated an EBOV stock that is enriched for DVGs by passaging the virus at a high MOI. RNA sequencing of A549 cells infected with either a high-DVG stock (HD-EBOV) or an EBOV stock without detectable DVGs (LD-EBOV) revealed a large number of differentially expressed genes. Infection with HD-EBOV resulted in substantial activation of immune signaling pathways, resulting in increased expression of proinflammatory cytokines and chemokines. This upregulation of antiviral factors was detected in A549 cells and differentiated THP-1 cells by qRT-PCR and multiplex immunoassay, starting from 8 hours post-infection. In addition, lower quantities of viral RNA and infectious virus were detected in HD-EBOV infected cells compared to LD-EBOV infected cells, suggesting interference with full-length genome replication. Further study will lead to a better understanding of EBOV pathogenesis and how these how these DVGs impact the innate immune response during EBOV infection.