Marseillevirus is a type of giant virus that infects Acanthamoeba castellanii. Like other giant viruses, Marseillevirus has a large genome and encodes several potential protein translation factors. Genomic analysis revealed that Marseillevirus Shanghai strain (Mar-SH2016) encodes four potential translation factors, one of which, ORF314 gene, has a high homology (60%) with the eukaryotic translation initiation factor eIF2/eIF5. But the role of ORF314 in virus replication remains to be revealed. In this study, three pairs of small interfering RNA (siRNAs) were designed to target ORF314 gene. Transfection of amoeba cells with fluorescence-labeled siRNAs showed that about one third of the Acanthamoeba castellanii cells were successfully transfected. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting confirmed that the siRNAs decreased the mRNA level of ORF314, and the results of virus titration showed that the level of virus replication decreased significantly (P<0.01). It is indicated that the potential translation initiation factor ORF314 of Mar-SH2016 has a role in the replication of the virus.