The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labelling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light into the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acid biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acid biosynthesis in procyclic T. brucei.