Pathogenic trypanosomatids have a large number of protein kinases and phosphatases in comparison to other organisms. Moreover, post-translational modifications have key role in the gene expression control in these cells, reinforcing the relevance of the phosphorylation process. Nevertheless little is known about protein phosphorylation in these protozoa. In front of this, the effects of the depletion of a MAP kinase-like kinase (MAPKLK1) were evaluated in Trypanosoma brucei. After silencing MAPKLK1 expression by RNAi, the cells were evaluated by SILAC MS-based proteomics. In total, 1,756 phosphorylation sites were identified, of which 384 were not previously described in T. brucei. The modulations observed on proteome and phosphoproteome are related to key cellular processes enriched to mRNA processing and stability control.