Paratuberculosis is a chronic intestinal infection in ruminants, caused by Mycobacterium avium subspecies paratuberculosis (Map). To study the role of host genetics in disease susceptibility, the Toll-like receptor 2 (TLR2) gene, selected based on its potential role in immunity to mycobacterial infections, was analyzed for single nucleotide polymorphisms (SNP) and their potential association with disease. For SNP discovery and to study SNP association with disease, a case-control study including 24 cows from farms with paratuberculosis was conducted. Sequence analysis of the TLR2 genes from 12 paratuberculosis-infected animals and 12 age-matched healthy herd mates revealed 21 different SNP. The TLR2-1903 T/C SNP was significantly associated with resistance to Map. This and four additional TLR2 SNP were studied in a subsequent observational field study with 553 cows from farms with paratuberculosis. The allelic distribution of the TLR2-1903 T/C SNP was confirmed to be significantly different between the infected and non-infected animals. For the TLR2-1903 T/C SNP the odds ratio was calculated, and similar to the dominance model in the association study, the CT and CC genotypes were compared to the TT genotype. Cows with the TLR2-1903 T/C mutation (i.e., the CT and CC genotypes) were at 1.7 (95% CI: 1.2, 2.8) times the odds of being Map-infected compared to cows with the TT genotype. In in vitro functional assays, monocyte-derived macrophages from animals with a TLR2-1903 TT genotype produced more IL12p40 and IL1beta when stimulated with Map compared to cells derived from TLR2-1903 CT and CC genotypes. Also, T cell proliferative responses to mycobacterial antigens were higher in animals with a TLR2-1903 TT genotype. In conclusion, we have found a significant association between SNP TLR2-1903 T/C in the bovine TLR2 gene and bovine paratuberculosis infection. This SNP and other genetic markers could be useful in marker-assisted breeding strategies as an additional tool in paratuberculosis control strategies. In addition, the functional studies suggest that genetic polymorphisms in bovine TLR2 which result in higher macrophage activity may contribute to enhanced T cell activation and a lower susceptibility to paratuberculosis in cattle.