Macrophage migration inhibitory factor (MIF) is a unique pro-inflammatory cytokine whose chief functions include modulating TLR4 expression, and suppressing the anti-inflammatory effects of glucocorticoids. Not surprisingly, MIF is involved in a number of inflammatory diseases and single nucleotide polymorphisms (SNPs) have been implicated in modulating disease severity. The objective of the present study was to determine if SNPs in 5' region of bovine MIF affects its promoter activity. Three SNPs were identified, -1078A>G, -395A>G, and -400G>C, all of which fall within predicted transcription factor binding regions. Reporter gene assays indicate that the identified SNPs have a significant effect of modulating MIF promoter activity. Finally, gene association analysis suggests a significant relationship of -395A>G with the susceptibility to Mycobacterium avium ssp. paratuberculosis infection, the causative agent of Johne's disease. Given the relationships revealed in the current study, it is clear that the role of MIF in bovine diseases such as Johne's disease merits further investigation.