Cold shock proteins are members of a family of DNA- and RNA-binding proteins with one or more evolutionarily conserved cold shock domain (CSD). These proteins have a wide variety of biological functions, including DNA-damage repair, mRNA stability, and regulation of transcription, splicing and translation. We previously identified two CSD containing proteins, CSD1 and CSD2, in the protozoan parasiteTrypanosoma bruceito be required for RBP6-driven metacyclic production, albeit at different steps of the developmental program. During metacyclogenesisT.bruceiundergoes major morphological and metabolic changes that culminate in the establishment of quiescent metacyclic parasites and the acquisition of mammalian infectivity. To investigate the specific role of CSD1 and CSD2 in this process, we ectopically expressed CSD1 or CSD2 in non-infectious procyclic parasites and discovered that each protein is sufficient to produce infectious metacyclic parasites in 24 hours. Domain truncation assays determined that the N-terminal domain, but not the C-terminal domain, of CSD1 and CSD2 was required for metacyclic development. Furthermore, conserved amino acid residues in the CSD of CSD1 and CSD2, known to be important for binding nucleic acids, were found to be necessary for metacyclic production. Using single-end enhanced crosslinking and immunoprecipitation (seCLIP) we identified the specific binding motif of CSD1 and CSD2 as “ANACAU” and the bound mRNAs were enriched for biological processes, including lipid metabolism, microtubule-based movement and nucleocytoplasmic transport that are likely involved in the transition to bloodstream form-like cells.