Background: Bacteria are capable of responding to various stressors, something which has been essential for their adaptation, evolution, and colonization of a wide range of environments. Of the many stressors affecting bacteria, we can highlight heavy metals, and amongst these, copper stands out for its great antibacterial capacity. Using Mycobacterium tuberculosis (Mtb) as a model, the action of proteins involved in copper homeostasis has been put forward as an explanation for the tolerance or adaptive response of this mycobacteria to the toxic action of copper. Therefore, the aim of this study was to confirm the presence and evaluate the expression of genes involved in copper homeostasis at the transcriptional level after challenging Mycobacterium avium subsp. paratuberculoisis (MAP) with copper ions. Methodology: Buffer inoculated with MAP was treated with two stressors, the presence of copper homeostasis genes was confirmed by bioinformatics and genomic analysis, and the response of these genes to the stressors was evaluated by gene expression analysis, using qPCR and the comparative ΔΔCt method. Results: Through bioinformatics and genomic analysis, we found that copper homeostasis genes were present in the MAP genome and were overexpressed when treated with copper ions, which was not the case with H2O2 treatment. Conclusion: These results suggest that genes in MAP that code for proteins involved in copper homeostasis trigger an adaptive response to copper ions.