eIF4A is a RNA helicase considered the prototype for the DEAD-box family and it is required in different events associated with the metabolism of RNAs, with the DEAD motif important to its ATPase and helicase activities. Previous studies have indicated that EIF4AI is the functional homolog working on protein synthesis in trypanosomatids. In order to investigate in more detail the interactome of the T. brucei EIF4AI in the translation context, as well as the importance of the DEAD motif for the main protein-protein interactions, we performed co-immunoprecipitation assays of HA-tagged EIF4AI and a version with a mutation on the DEAD motif (DQAD), combined to mass spectrometry analysis. The analysis confirmed a specific association of EIF4AI with an eIF4F complex based on EIF4E4/EIF4G3 subunits, and other translation factors. A differential pattern of association was seen for the mutant protein, indicating that the DEAD motif might be important to the specific eIF4F assembly in T. brucei. This study helped to clarify functional aspects of the EIF4AI helicase during Trypanosoma mRNA translation/metabolism.