The actinobacterium Streptomyces sp. MC1 has previously shown the capacity to resist and remove Cr(VI) from liquid culture media. The aim of this work is to analyze the differential expression pattern of intracellular proteins when Streptomyces sp. MC1 is exposed to Cr(VI) in order to explain the molecular mechanisms of resistance that this microorganism possesses. For this purpose, 2D-PAGE and shotgun proteomic analyses (2D-nanoUPLC-ESI-MS/MS) were applied. The presence of Cr(VI) induced the expression of proteins involved in molecular biosynthesis and energy generation, chaperones with a key role in the repair of misfolded proteins and stress response, transcription proteins, proteins of importance in the DNA supercoiling, repair and replication, and dehydrogenases involved in oxidation-reduction processes. These dehydrogenases can be associated with the reduction of Cr(VI) to Cr(III). The results of this study show that proteins from the groups mentioned before are important to face the stress caused by the Cr(VI) presence and help the microorganism to counteract the toxicity of the metal. The use of two proteomic approaches resulted in a larger number of peptides identified, which is also transduced in a significant number of protein ID. This decreased the potential complexity of the sample because of the protein dynamic range, as well as increased the recovery of peptides from the gel after digestion.