Macrophages pertain to the mononuclear phagocytes system as part of the innate and acquired immunity responses. The interaction of macrophages with Mycobacterium bovis concludes with the formation of granulomas. However, the host-immune response to M. bovis could be compromised by the suppression of immuno-regulatory pathways leading to active tuberculosis. Therefore, transcriptome analysis of the macrophage response to M. bovis can offer insight into the host-pathogen interaction. Bovine monocyte derived macrophage (MDM) of peripheral blood was obtained from 7 non-infected Holstein cows, cultivated and infected with M. bovis. The RNA was isolated 24 h after infection and single-end reads were mapped to the Bos taurus (UCSC_bosTau7) reference genome using Tophat v2.012. Using Cufflink v2.2.1, 7,664 transcripts from 7,505 genes were tested and 1,192 transcripts from 1,187 genes were differentially expressed between the infected and non-infected MDM (false discovery rate adjusted P-value <0.05). Among the transcripts, 61% were upregulated in the infected relative to non-infected MDM. Interferon gamma (IFNG), indoleamine 2,3-dioxygenase 1 (IDO1), chemokine (C-X-C motif) ligand 10 (CXCL10), and chemokine (C-X-C motif) ligand 9 (CXCL9) were overexpressed in infected relative to non-infected MDM. The IFNG is a potent activator of macrophages, while IDO1 encoded an important rate-limiting enzyme in the kynurenine pathway, activated during the inflammation process that favors immune suppression and tolerance. The CXCL9 and CXCL10 are critical during the early stages of the immune response to M. bovis. The top 5 ranking category clusters detected by the functional analysis of the differentially expressed genes using DAVID (enrichment score >5) included immune defense, and the activation and proliferation of leukocyte, lymphocyte, mononuclear cells, and T and B cells as well as cytokines production and apoptosis regulation. This study unveils details of the host response of MDM to M. bovis.