Background: Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic viral disease that is asymptomatic in infected animals, but a serious threat to humans. Ticks of the genus Hyalomma seem to be the principal vectors and they play an important role as a natural reservoir. CCHFV establishes a persistent infection in ticks and they can transmit the virus to their hosts during blood feeding. The coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. It seems to play a role during viral genome replication and the apoptosis process in mammalian cells. Methods: The CCHFV strain IbAr 10200 (wtCCHFV) and a recombinant CCHFV (rCCHFV), containing the mutated sequence AEVA instead of the DEVD domain, were used to evaluate the effects of the DEVD domain mutation on CCHFV infection in mammalian and tick cell lines. Results: Experiments showed that the rCCHFV replicates less efficiently than the wtCCHFV in mammalian and tick cell lines. In particular, as shown by virus titration and PCR assay, the wtCCHFV was able to establish a persistent infection in tick cells while the rCCHFV decreased over time and the cells were negative for the presence of the virus at 127 days post infection. Conclusion: Considering that the DEVD domain is conserved in all CCHFV strains, it appears to be essential for efficient viral replication in target cells and for the establishment of persistent infection in ticks.