Mycobacterium bovis (M. bovis) is a pathogenic bacteria that generates approximately $3 billion worldwide production loss for the cattle industry, in terms of animal value and productivity. To help understand the response to this pathogen, the objective of this study was to characterize the transcriptome of cows infected with M. bovis relative to healthy controls. Cows positive for M. bovis were identified using the single intradermal comparative tuberculin test, and in vitro ELISA-based interferon-gamma. The transcriptome of peripheral blood leukocytes from Holstein cows detected positive was compared with healthy control cows (n = 8/group) using individual RNA-Seq libraries. Single-end reads were mapped to the Bos taurus reference genome (UCSC_bosTau7) using Tophat v2.0.12. In total, 8,309 isoform transcripts pertaining to 8,127 genes were identified and 2,620 isoform transcripts pertaining to 2,608 genes were identified as differentially expressed (False Discovery Rate-adjusted P-value <0.05) using Cufflinks v2.2.1. Among the top 25 differentially abundant transcripts using log fold change, 76% were overexpressed in the infected group relative to control cows including polo-like kinase 3 (PLK3), atypical chemokine receptor 3 (CXCR7), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor delta (NFKBID). PLK3 participates in the tuberculosis response pathway, activating the nuclear factor-κB, which in turn stimulates transcription of the inducible nitric oxide synthase as well as pro-inflammatory cytokines. CXCR7 is an innate immune gene reported in leukocytes infected with M. bovis. Also, NFKBID may regulate the expression of cytokines by regulating nuclear factor-κB activity. Functional analysis of the differentially expressed genes using DAVID identified 14 enriched functional category clusters (enrichment score >3) including leukocyte mediated immunity, leukocyte activation and proliferation, cellular apoptosis, together with cytokines and chemokines production. The majority of the genes in these clusters were overexpressed in the infected group relative to control cows. These results offer insights on leukocyte transcriptome changes in response to M. bovis infection.