AbstractmRNAs lacking 5′ untranslated regions (leaderless mRNAs) are molecular relics of an ancient translation initiation pathway. Nevertheless, they still represent a significant portion of transcriptome in some taxons, including a number of eukaryotic species. In bacteria and archaea, the leaderless mRNAs can bind non-dissociated 70 S ribosomes and initiate translation without protein initiation factors involved. Here we use the Fleeting mRNA Transfection technique (FLERT) to show that translation of a leaderless reporter mRNA is resistant to conditions when eIF2 and eIF4F, two key eukaryotic translation initiation factors, are inactivated in mammalian cells. We report an unconventional translation initiation pathway utilized by the leaderless mRNA in vitro, in addition to the previously described 80S-, eIF2-, or eIF2D-mediated modes. This mechanism is a bacterial-like eIF5B/IF2-assisted initiation that has only been reported for hepatitis C virus-like internal ribosome entry sites (IRESs). Therefore, the leaderless mRNA is able to take any of four different translation initiation pathways in eukaryotes.