Proliferative stage (tachyzoite) is a critical factor in the transmission and pathogenesis of Toxoplasma gondii (T. gondii), which is capable of infecting a large variety of host cells. Proto-oncogene eukaryotic translation initiation factor (eIF-5A) is thought to function in regulation of many cellular processes including cell proliferation. Investigation the specific roles of eIF-5A involved in T. gondii is instrumental for the elucidation of tachyzoite replication. Here, eIF-5A knockdown strain was generated and analyzed by isobaric tags for relative and absolute quantification (iTraq), which suggested that proto-oncogene eIF-5A also acts as an essential gene to ensure proper protein synthesis during T. gondii invasion and replication.